DEVELOPING AND EVALUATING PERENNIAL ORNAMENTALS AND TEMPERATURE-TOLERANT ORCHID HYBRIDS FOR DURABILITY AND RESILIENCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 1011239
• Project Start Date: Nov 1, 2016
• Project End Date: Sep 30, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
KENTUCKY STATE UNIVERSITY
(N/A)
FRANKFORT,KY 40601

Performing Department
Agriculture & Environmental Science

Non Technical Summary
Kentucky is a state which has a relatively small industry for the production of nursery, greenhouse and floriculture crops, ranked at 41st. In 2012, the total sale for these crops, excluding Christmas trees but including sod was $ 79,283,000 substantially smaller than that of neighboring states such as Indiana ($110,838,000), Ohio ($437,723,000), and Tennessee ($294,740,000). One strategy to increase profitability for greenhouses and nurseries is to specialize in niche crops with a high value.The 2015 wholesale value of floriculture crops increased 4 percent from the valuation of the previous year. This increase was the highest in the last 10 years, marking an important trend as the U.S. economy is back on track for growth. According to the USDA data, 46 percent of U.S. consumers purchased outdoor ornamental and nursery plants, 34 percent cut flowers and 20 percent indoor houseplants. Of all potted plants, orchids continue to lead this category, with $288 million in value, up 5 percent from 2014, followed by poinsettias ($140 million). Orchids have been considered a niche crop in the past, but are rapidly gaining popularity. Since the whole wholesale orchid sales were over $186 million in 15 Program States in 2012, a substantial increase of over $100 million was observed in just three years. Despite their popularity, production of orchids is heavily concentrated in three states: California, Florida and Hawai'i. In contrast, some of once prominent orchid production areas such as Chicago and the Great Lakes area have been decimated. As orchids require a considerable amount of heat for optimum growth, the rising heat cost is a dire concern for orchid growers. Development of orchids which not only require less heat for production, but also can be potentially grown in high tunnels or even gardens in the USDA Plant Cold Hardiness Zone 6 or 7 will create opportunities for nurserymen and limited resource farmers as well as hobbyists and consumers.Aside from tropical indoor houseplants, consumers are interested in hardy ornamentals that are durable and resilient in their gardens. The ideal candidates for this project are Begonia, Helianthus and Zinnia. This project includes genetic studies, which are aimed to impart cold hardiness and perenniality by interspecific hybridization and subsequent induced polyploidy with chemical agents.

Animal Health Component

80%

Research Effort Categories

Basic

20%

Applied

80%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
202	2122	1081	70%
203	2122	1081	30%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 202 - Plant Genetic Resources;

Subject Of Investigation
2122 - Potted plants;

Field Of Science
1081 - Breeding;

Keywords

orchids

begonia

helianthus

zinnia

perenniality

cold-hardiness

polyploids

Goals / Objectives
The over all goalsfor this project are to develop cold-hardy or cold temperature tolerant ornamentals, such as orchids and Begonia, and introduce perenniality into Helianthus and Zinnia. Dendrobium orchids that can be successfully grown with a lower energy input such as cold frame and high tunnels. Specifically, temperature tolerance will be introduced through the use of cold-hardy or temperature tolerant orchid, Begonia, and Zinnia species and polyploidization with antimicrotubule agents such as oryzalin. Perenniality into common garden plants will be introduced by interspecific hybridization with perennial counter parts. Research progress and findings will be disseminated through publication of peer-reviewed scientific journal articles, presentation at both national and regional scientific meetings, Kentucky State University's Extension activities, presentations at regional plant society meetings as well as the use of social networking and/or media.

Project Methods
Objective 1. Impart cold hardiness into three orchid groups and Begonia through interspecific hybridization and polyploidization.Stock plantsOrchid species and hybrids have been acquired for the previous Evans-Allen project. These plants will continue to be used along with newly acquired plants. Begonia, seeds and plants will be newly acquired from both commercial and The U.S. National Plant Germplasm System.Plant cultureAfter purchase, plants will be maintained in the greenhouse at KSU Research Farm. Upon arrival, plants will be inspected for disease and pests. Plants will be provided ample water and regular fertilization with Michigan State University formulated fertilizer (13-3-15 8Ca-2Mg) during active growth. Winter temperature will be maintained above 15?C. As Cymbidium, Dendrobium, and Laeliinae species require moderate light for optimum growth, the greenhouse will be shaded (30~50%%) during summer.Pollination and seed harvestingPollen of parents will be collected from two to three-day-old flowers if the pod parent is not available. Pollen will be stored in microfuge tubes enclosed with silica gel at -20?C. Pollen mass will be attached to stigma of pod parent with a tooth pick and then pushed a little. Jewelry tags will be used to record parentage of the cross, which will also be recorded electronically. Special attention will be paid to plants with developing seed capsules since environmental shock or stress can cause plants to drop them.Medium preparationTwenty two grams of Vacin and Went medium basal salt (1949) will be added to approximately 900 ml of double distilled water (DDH2O). The pH of the medium will be adjusted to 5.3 prior to auctoclaving. The final volume will be then adjusted and 7.0g of agar will be added before heating the mixture on a hot plate. Medium will be dispensed into a Magenta™ vessel GA-7 Plant Culture Box, and autoclaved with a standard condition (at 121?C and 100 kPa and for 20 min.)Seed viability, and seed and seedling cultureAfter harvesting seed capsules, they will be briefly submerged into 100% ethanol, and then flamed in a laminar flow. Seed capsules will be surface sterilized with 10% commercial bleach for 10 min., and placed in a sterile Petri dish. A surgical scalpel and forceps will be used to remove a part of a seed capsule, and seeds will de sown directly to Vacin and Went medium with the scalpel. If seeds are not dispersed evenly, aliquot of sterile double distilled water will be added. Cultural vessels will be placed under fluorescent light at 10 μmol m-2 s-1 for 16 hrs/day. Ambient temperature will be maintained at 20?C. Seedlings will be subcultured every three months, depending on their growth.A portion of seeds (100~250) will be saved during seed sowing. These seeds will be stained with fluorescein diacetate (Woods et al, 2006). FDA will be prepared at 0.5% (w/v) in absolute acetone (Widholm, 1972) and mixed, on a slide, 1:1 (v/v) with seeds in distilled water. Stain development will be observed up to 24 hrs. to determine optimum duration. Staining will be viewed with an inverted microscope with UV light.Additionally, experiments will be conducted to test different medium substrate for growth optimization using agar, carrageenan, gellan gum, and liquid (substrate-free) medium. Weight increase, shoot number, as well as survival rate after acclimatization will be recorded for each treatment. At the end of the project period, the following measurements will be taken for each grex: height, weight, the number of shoots, and leaf shape. Upon successful completion of this part of the project and continuation of the next phase, cold-hardiness of resulting hybrids will be tested and horticultural characteristics such as flower shape, flower size, the number of flowers per raceme, plant height at maturity, etc. will be recorded for each grex.Polyploidization Protocorms of hybrids will be subjected to three different concentrations of oryzalin 14.4, 28.9, and 57.7 μM (w/v) for five days, using a modified method of Miguel and Leonhardt (2011). Fifty protocorms will be transferred to a membrane raft in a Magenta culture vessel containing liquid Vacin and Went medium. Oryzalin will be first dissolved in Dimethyl sulfoxide and then diluted with methanol. After sterilizing the methanolic stock with a syringe filter, the appropriate amount of oryzalin solution will be added to the liquid medium. After five days, protocorms on raft membranes will be transferred to cultural vessels with liquid Vacin and Went medium. Three replications will be made for each grex and will be repeated two times. Survival rate of protocorms/seedlings will be determined after 3 months. Ploidy levels of those that survived will be analyzed with flow cytometry. The size of stomata will also be measured.Flow cytometric analysisLeaf tissue will be collected from seedlings (not protocorms) and will be cut into pieces with a razor blade in extraction buffer, containing followings: 100 mM MgCl2, 40 mM trisodium citrate, 22mM Mops, and 0,1% Triton X. The pH of the buffer will be adjusted to 7.1. Cell suspension will be filtered through a 50 mm mesh filter and stained with 500 ?l propidium iodide (Ascough et al., 2008). Fluoresce will be measured with Cell Lab QuantaTM SC Flow Cytometer (Beckman Coulter, Chicago). Untreated Den. moniliforme and Den. kingianum stock plants will be used as a reference to compare the DNA contents of oryzalin-treated seedlings.Objective 2. Introduce perenniality into Helianthus and Zinnia.Stock plantsVarious commercial H. annuus cultivars will be purchased from commercial sources. They will be started in May and August to use them as both pollen and seed parents. Perennial Helianthus species such as H. × laetiflorus, H. rigidus and H. tuberosus, and sterile perennial hybrids of H. annuus, including H. 'Happy Days' will be purchased from commercial nurseries and acquired from The U.S. National Plant Germplasm System if available. Seeds of Z. violacea and Z. grandiflora will be acquired from commercial sources.Pollination and acquisition of hybridsPollen of H. annuus and Zinnia species will be collected when they bloom and will be individually stored at -20?C until ready. Twenty min. after removal from the storage, pollen will be manually transferred to the disc flowers of perennial sunflowers. Seeds will be harvested at full maturity and will be sowed next spring.Embryo rescue will be carried out to acquire hybrids if necessary. Five day old embryos will be cultured on Gamborg's B5 salts medium (Gamborg et al., 1968) supplemented with vitamins, amino acids, α-naphthalene acetic acid), and 120 g/l sucrose as described by Feng et al. (2006). Subsequent growth of embryo and germination will take place on the same medium with at reduced sucrose content (20g/l).Polyploidization? Callus of confirmed Zinnia hybrids will be initiated on MS medium with BA and 2,4-D. Callus will be subjected to three different concentrations of oryzalin 14.4, 28.9, and 57.7 μM (w/v) for five days, using a modified method of Miguel and Leonhardt (2011). Regenerating shoots will be cultured and maintained on a same medium without any PGR's.Flow cytometric analysisLeaf tissue will be collected from shoots in culture and will be analyzed as described above.Objective 3. Evaluate cold hardiness and tolerance of hybridsCold hardiness of hybrids will be tested in the greenhouse at the KSU Harold R. Benson Research and Demonstration Farm and High tunnels and demonstration gardens on the KSU Campus. Orchid hybrid seedlings will be maintained in heated (5?C minimum) and unheated sections of the high tunnels and greenhouses (-5?C minimum). Cold hardiness of Begonia, Helianthus and Zinnia hybrids will be tested on campus and research farm demonstration gardens. Cooperators of the project will also test cold hardiness or temperature tolerance of hybrids from this project.

Progress 11/01/16 to 09/30/20

Outputs
Target Audience:The target audience for this project is professional and amateur orchid and Begonia growers and growers of ornamental plants in Kentucky and surrounding areas. The PI has disseminated information about the project through orchid shows in Louisville, KY, Cincinnati, OH, Dayton, OH, and Strongsville, OH, as well as monthly judging meetings at the American Orchid Society Cincinnati Judging Center in Cincinnati, OH. Additionally, the PI has been regularly making contact with growers of ornamental plants, including Tony Avent (Plant Delights Nursery), Russ Vernon (New Visions Orchids), Wayne Ferrell (Santa Barbara Orchid Estate), Darren Heppel (Steve's Leaves) and Darrin Norton (Mountain Orchids) regarding the project. The PI has also disseminated germplasm materials to the project collaborators and also to the Ornamental Plant Germplasm Center at The Ohio State University. Changes/Problems:No major change was made during this period. The COVID pandemic had an impact on aquisition of materials, access to the farm and research lab, as well as social distancing and self-quarantine made the project more challenging. What opportunities for training and professional development has the project provided?This project has provided various training and development opportunities to the PI and project participants. The PD has been a orchid judge with American Society, and has attended monthly judging held in Cincinnati, Oh, and attended regional shows as an associate and later as a fully accredited judge. The PD has also exhibited the orchids at the regional show to exemplify and disseminate information regarding the current project.The PD served as chairs for Orchids Professional Interest Group, and Tropical Horticulture Professional Interest Group with American Society for Horticultural Science. The PD proposed workshops and professional tours for the PIGs and ASHS members. The PD also worked with Dr. Marion Simon for demonstration of orchid growing and plant propagation through her The Third Thursday Program. In addition, the PD participated in Kentucky State University's Summer program, and high school students learned plant propagation and orchid mounting. How have the results been disseminated to communities of interest?Information regarding the project progress has been disseminated at both local, regional, and national scientific meetings and conferences, including those of American Society for Horticultural Science, American Society for Horticultural Science Southern Region, Kentucky Academy of Science, andAssociation of 1890 Research Directors Research Symposium. In addition, the PD communicated with the project collaborators on the research progress and also disseminated germplasm materials acquired in this project. Finally, the PD disseminated information through monthly judging and regional shows he attended to reach both commercial and armature orchid growers.? What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Helianthus and Zinnia breeding has been dropped previously because of space limitation and the lack of success. One hundred forty eightcrosspollinations of orchids, including Cattleya types (107), Cymbidium (6), Dendrobium (12), and other orchids (23) were made during this reporting period. Initiation of orchid seedling is hampered by the lack of adequate facility as the research bulding was not complete. Despite the circumstance, seedlings of ten orchid crosses have been initiated in vitro, and will soon be cultured under the greenhouse environment after acclimitization.Additionally, 256 cross pollinations were made forBegonia. germination tests have been conducted and some of seedlings have been grown to maturity. One clone of B. formosana x B. grandis 'Heron's Pirouette,' two clones of B. grandis 'Heron's Pirouette' x B. U358, one clone of B. grandis 'Heron's Pirouette' x B. U400, and several clones of B. grandis 'Heron's Pirouette' x B.ningmingensis var. bella have been selected for overall desirable horticultural characteristics. Seedlings of other crosses such as B. grandis 'Sapporo' x B. muliensis and B. grandis 'Heron's Pirouette' x B. picta have been acquired, but they have not reached to maturity yet. These will be evaluated for cold-hardiness and bred to further impart temperature-tolerace.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Kobayashi, H. 2020. Orchids as medicines. Presented at the annual meeting of American Society for Horticultural Science

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audience for this project is professional and amateur orchid and Begonia growers and growers of ornamental plants in Kentucky and surrounding areas. The PI has disseminated information about the project through orchid shows in Cincinnati, OH as well as monthly judging meetings at the American Orchid Society Cincinnati Judging Center in Cincinnati, OH. Additionally, the PI has been regularly making contact with growers of ornamental plants, including Tony Avent (Plant Delights Nursery) and Wayne Ferrell (Santa Barbara Orchid Estate) and Darrin Norton (Mountain Orchids) regarding the project. Changes/Problems:Low rate of the seed set andtime required to reach full seed maturity are two ofmajor issues for orchid cross pollination. In the case of Begonia breeding, some of them are reluctant to bloom in the current greenhouse conditions. In addition, some of them are very prone to die back, and never recover. Improvement in the fertilization program has resulted better blooming and performance. What opportunities for training and professional development has the project provided?The PI was able to attend two conferences as well as the American Orchid Society member meeting in San Diego, California. The PI also developed the tour proposal and workshop proposal for the ORCHID Professional Interest Group (PIG) and TROP PIG within American Society for Horticultural Science. How have the results been disseminated to communities of interest?Findings from the current project have been presented at the Annual Conference of American Society for Horticultural Science and 1890 Research Associate Directors Symposium. Additionally, progress of the project has been routinely discussed with members of regional orchid societies and orchid judges at the Cincinnati Judging Center. What do you plan to do during the next reporting period to accomplish the goals?The Begonia hybrids will be grown to maturity, and will be cross back toB. grandisto further impart cold hardiness. Acquired seedling from this process will be grown to maturity, then evaluated for their traits such as leaf variegation, height, cold-hardiness and overall performance.In addition, polyploidzation of selected seedlings will be attempted to expedite the breeding process and also potentially increase cold-hardiness. Orchid seedlings will also be grown to full maturity in order to facilitate breeding.

Impacts
What was accomplished under these goals? Collection of Begonia and orchid species and hybrid germplasm continues from various sources, including the Ornamental Plant Germplasm Center (OPGC) at The Ohio State University and a Begonia nursery in Japan. Some of the Begonia germplasm has been shared with the OPGC, and orchids with orchid growers. A total of 684 cross pollinations were made, mostly using B. grandis 'Heron's Pirouette' as a capsule parent. The pollen parents that produced seeds included, B. formosana albomaculata, B. pearcei, B. roxbughii,B. sp. cf. villifolia OJ10-VN140, B. U400, B. U485, and B. U489. Germination tests have been performed for Begonia seeds harvested, and findings from this portion of the study has been presented at two conferences.Similarly, 685 cross pollination of Cattleya, Cymbidium, Dendrobium andmiscellaneous genera were made during this reporting period. The low rate of successful fertilization leading to dehiscing as well as extensive time required before seed harvest is a persistent problem for orchid hybridization.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kobayashi, H. (2019). Imparting Cold-hardiness into Begonia through Interspecific Hybridization. Poster presented at the Annual American Society for Horticultural Science Conference in Las Vegas, NV.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kobayashi, H. (2019). Strategies for developing cold-hardy Begonia for the South. Oral presentation at Association of 1890 Research Directors Research Symposium 2019 in Jacksonville, FL.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience for this project is professional and amateur orchid and Begonia growers and growers of ornamental plants in Kentucky and surrounding areas. The PI disseminated information about the project through orchid shows in Louisville, KY, Cincinnati, OH, and Santa Barbara, CA, as well as monthly judging meetings at the American Orchid Society Cincinnati Judging Center in Cincinnati, OH. Additionally, the PI has been regularly making contact with growers of ornamental plants regarding the project. Changes/Problems:While the following problems have been properly addressed, there were three instances that the facility issues have substantially impacted the outcome of the project. On Nov, 16, 2017, the greenhouse heater was found to be broken, eliciting some orchid plants to abort seed capsules. On Dec. 11, 2017, thermostats in both two greenhouses failed and approximately 70% of orchids were killed or severely damaged in one of the greenhouses. Furthermore, extreme heat (115°F) during the renovation of the greenhouse complex further affected orchids, resulting in abortion of almost all seed capsules. Very scarce flowering was observed during these periods. Recovery of orchids has been slow, which has substantially affected the outcome of the project. Due to limitations on the greenhouse space available, breeding efforts will be concentrated on Begonia and orchids. What opportunities for training and professional development has the project provided?I have organized a professional tour at the annual conference of the American Society for Horticultural Science in Washington, DC. Additionally, I have participated in orchid judging at the Miami Valley Orchid Show (Feb. 17&18), Santa Barbara Orchid Show (March 16 to 19, 2018), and Greater Cincinnati Orchid Society Fall Show (Oct. 20&21). I visited a collaborator site in Santa Barbara to discuss the progress of the project and visited the Ornamental Plant Germplasm Center (Columbus, OH). How have the results been disseminated to communities of interest?Research findings and progress have been disseminated through presentations at the American Society for Horticultural Science conference, Kentucky Academy of Science meeting, and various orchid shows. What do you plan to do during the next reporting period to accomplish the goals?Acquired Begonia seedlings will be first selected for their aesthetic values and performance. These selected seedlings will be tested for their cold-hardiness, and will also be used as the parents to increase cold-hardiness of hybrids further. If sterility becomes an issue for some of clones, polyploidization will be attempted.

Impacts
What was accomplished under these goals? In this reporting period, 265 orchid crosses and 1031 begonia crosses were made. Most of the Begonia crosses were between distantly related species or species and hybrids aborted prematurely, with exceptions of B. grandis 'Heron's Pirouette' x B. 'Non-Stop Salmon,' B. grandis 'Heron's Pirouette' x B. 'Encanta Orange,' B. grandis 'Heron's Pirouette' x B. 'Encanta Pink,' B. grandis 'Heron's Pirouette' x B. dregei. Germination tests were performed with seeds of some of crosses. Of ten crosses that were tested, only seeds from three crosses germinated. Those were B. formosana x B. grandis 'Heron's Pirouette' (4%), B. formosana x B. 'Chandler's Hardy' (60%), and B. grandis 'Sapporo' x B. muliensis (16%). Additionally, seedlings of B. grandis 'Heron's Pirouette x B. ningmiensis var. bella were acquired. None of the seeds from the following crosses have germinated: B. emeiensis x B. 'Chandler's Hardy,' B. grandis 'Heron's Pirouette' x B. dregei, B. 'Non-Stop White' x B. grandis, B. 'U400' x B. 'Smooch,' B. rex x B. 'Smooch,' 'B. Smooch' x B. formosana, B. 'Smooch' x B. grandis 'Heron's Pirouette.' Acquisition of orchid and Begonia germplasm continues. I have received 14 Begonia accessions from the Ornamental Plant Germplasm Center (Columbus, OH) and four accessions from the JC Raulston Arboretum (Raleigh, NC). I have also donated 5 Begonia accessions to the JC Raulston Arboretum, four Begonia accessions to Plant Delights Nursery (Raleigh, NC) and one to the Ornamental Plant Germplasm Center. I have also donated six orchid accessions to Santa Barbara Orchid Estate (Santa Barbara, CA).

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Kobayashi, H. Developing cold-hardy Begonia hybrids for Kentucky. Presented at Kentucky Academy of Science Annual Meeting, Murray State University, Murray, KY, November 3-4, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Kobayashi, H. Strategies for developing cold tolerant epiphytic orchids. Presented at the American Society for Horticultural Science Conference, Washington, DC, August 1-3, 2018.

Progress 11/01/16 to 09/30/17

Outputs
Target Audience:The target audience for this project is professional and amateur orchid and begonia growers and growers of ornamental plants in Kentucky and surrounding areas. The PI disseminated information about the project through orchid shows in Louisville, KY, Cincinnati, OH, and Strongsville, OH, as well as monthly judging meetings at the American Orchid Society Cincinnati Judging Center. Changes/Problems:Due to extreme temperature conditions in the greenhouse during the summer and winter, the condition of plants have been negatively affected, occasionally resulting in abortion of seed capsules or, in some cases, plant death. Creation of intergeneric hybrids was not difficult for Cattleya type orchids, but intersectional hybridization was difficult for both Cymbidium and Dendrobium. Persistence of western flower thrips (Frankliniella occidentalis) was another problem that affected the outcome of the project. Finally, the absence of appropriate research facilities due to renovations is a major culprit. What opportunities for training and professional development has the project provided?A presentation on Phragmipedium boissierianum prebreeding was made to American Orchid Society's judges at the Cincinnati Judging Center to acquaint them with concepts on breeding such as prebreeding, line breeding, and outcrossing. Additionally, two workshops on growing orchids (i.e., dividing and mounting) were presented at Kentucky State University's Third Thursday Thing Sustainable Agriculture Workshop series. I also organized a tour for the American Society for Horticultural Science, including visits to Pu'u'ala Farm and Ranch Inc. (kava and noni), Carmela Orchids (orchids), Onomea Tea Company (green tea), and Leilani Nepenthes (Asian pitcher plants). How have the results been disseminated to communities of interest?During this period, two presentations were made at the Kentucky Academy of Science and the Association of 1890 Research Directors. In addition, two presentations on polyploidy of L. anceps complex and breeding were presented at the annual conference of American Society for Horticultural Science and during a monthly judging meeting of the American Orchid Society at the Cincinnati Judging Center. What do you plan to do during the next reporting period to accomplish the goals?Acquisition of plant material and cross-pollination of orchids will continue. Seeds will be harvested upon maturation and seedlings will be started in vitro. With appropriate conditions and settings, protocorms will be subjected to antimicrotubule agents, such as oryzalin and colchicine to double the chromosome number. Similarly, acquisition of begonia germplasm will continue and seeds of interspeficic crosses will be germinated. B. 'Smooch', a purported hybrid between B. grandis and B. chitoensis, will be introduced in vitro. B. 'Smooch' will then be subjected antimicrotubule agents to acquire polyploids with regained fertility. Seeds of H. annuus will be started next spring and flowers will be pollinated with pollen from perennial sunflowers. Flowers will be emasculated with GA5 prior to pollination. Additionally, two cultivars of H. x multiflorus will be introduced into tissue culture. Callus will be generated and will be subjected to antimicrotubule agents to double the chromosome number.

Impacts
What was accomplished under these goals? A total of 290 cross-pollinations were made for Cattleya type orchids, including Epidendrum and Encyclia hybrids, 39 cross-pollinations for Dendrobium orchids, 31 cross-pollinations for Cymbidium orchids, and 42 cross-pollinations for others. Due to compatibility and adverse effects of the growing conditions/extreme temperatures in the greenhouse, the rate of successful pollination was low. For example, 28.3% of Cattleya-type crosses resulted in development of seeds capsules, and all of Cymbidium seed capsules eventually aborted. Subsequently, capsules from twenty-one Cattleya-type crosses eventually aborted or prematurely dehisced. Seeds were harvested from eighteen crosses during this reporting period, including crosses made last year. Seeds of twelve crosses were sown on plant tissue culture medium containing Hyponex medium (3 g/L), sucrose (20 g/L) and agar (8 g/L). Germination and subsequent development of protocorms were observed for four crosses. Three crosses of begnoia were made in the fall of 2016. Although a seed capsule of one cross (Begonia rex x B. grandis) developed, it was lost when the greenhouse temperature dropped below freezing. Twenty-two crosses of begonia, using B. grandis as both male and female parents and plants developed from cuttings provided by The Ornamental Germplasm Center (OGC) at The Ohio State University, were made during Oct. 2016 to Sept. 2017. Cuttings of twenty begonia species and seeds of two strains of B. grandis var. sinensis were received from OGC in May 2017. In return, cuttings of twenty-eight species and two hybrids were donated to the OGC. Accessions of 14 Helianthus species were acquired from National Plant Germplasm System (NPGS), whereas seeds of four species (H. giganteus, H. grosseserratus, H. maximiliani, and H. strumosus) and plants of two species (H. hirstus and H. tuberosus) and three perennial hybrids (H. x laetiflorus 'Lemon Queen', H. x multiflorus 'Capenoch Star', and H. x multiflorus 'Flore Pleno') were obtained from commercial nurseries. Pollen of perennial sunflowers were collected for hybridization with diploid H. annnus for next spring. Four accessions of Zinnia acerosa were acquired from NPGS along with seeds from mixed localities from Arizona (Batamote Germplasm desert zinnia). Pollination was made with pollen of Zinnia elegans cultivars (e.g., 'Oklahoma' mix and 'Cactus') onto Z. acerosa and Echinacea purpurea 'White Swan'. Embryos were rescued after 10 days and placed on Gamborg B5 medium as described for Helianthus. However, none of these embryos subsequently developed into seedlings.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Kobayashi, H. 2017. Strategies for developing cold tolerant epiphytic orchids. Presented at Annual Conference of American Society for Horticultural Science, Waikoloa, HI, September 19-22, 2017.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Kobayashi, H. 2017. Orchid breeding for cold tolerance at Kentucky State University. Presented at The Association of 1890 Research Directors Biennial Meeting, Atlanta, GA, April 1-4, 2017.