Cornus florida (flowering dogwood) is a valuable tree native to eastern North America and prized for its floral bracts and colorful foliage. However, the tree is highly susceptible to powdery mildew (PM), a common fungal disease that challenges ornamental plant production. There are bioengineering approaches to developing PM resistance that involve the introduction of genes into C. florida cells and the regeneration of plants through somatic embryogenesis (SE). SE is a process by which somatic cells have the capacity to produce embryos without sexual reproduction. In C. florida, the regeneration of transgenic somatic embryos into plants has been problematic. Our work aims to determine the impact of cold treatments on the germination of somatic embryos. We propose that short-term low-temperature treatments will improve embryo germination, considering past research has demonstrated the importance of periodic low temperatures on natural seed germination in woody plant species such as fruit trees. We cultured a transgenic line of C. florida embryogenic callus expressing a visual marker (ß-glucuronidase) and enriched for globular stage embryos. We then introduced these globular embryos into liquid suspension media allowing the embryos to proliferate pro-embryogenic masses (PEMs) needed for mass embryo production. We chose somatic embryos morphologically identical to zygotic embryos of the same stage of development for testing plant regeneration following exposure to four different temperature conditions over four different time periods. The four different temperatures included: (1) 3°C; (2) 4°C; (3) 7°C; and (4) 23°C as the control temperature. The four different time exposures to the different cold periods included 0, 2, 4, and 6 weeks. Following cold exposure for a designated time, we transferred the somatic embryos to germination media, exposing the embryos to fluorescent light at room temperature ( /-) 23°C. Successful germination of the somatic embryos was indicated by taproot elongation with the production of roots, greening of the cotyledons, emergence of the apical shoot, followed by expansion of epicotyl and primary leaves. This research will yield the first transgenic C. florida plants and enable the introduction of PM resistance using bioengineering methods.
