Tissue Culture Clonal Propagation of Hybrid White Oaks for the Urban Environment

A tissue culture system was developed to clonally propagate a series of hybrid white oaks (Quercus L.) at the plant breeding program of the Urban Horticulture Institute (UHI), Cornell University. From 2014 through 2018, 34 genotypes and 1966 individual explants of UHI hybrid white oaks were trialed to determine their capacity to establish, multiply, and root in a tissue culture environment. UHI hybrid oak genotypes were selected based on their known tolerance to stresses common in the urban environment (drought, alkaline soils, pests, and diseases) and their ornamental qualities. Individual genotype was the single largest factor affecting successful establishment, multiplication, stabilization, and rooting of hybrid oaks in vitro. Thirteen clones of hybrid oaks were identified as having the capacity to stabilize and grow continuously in the multiplication phase. Multiplication efficiency rates were dependent on individual genotype. Stabilized genotypes showed the capacity to be re-established during successive years. The tissue culture process was simplified and refined to make the protocols less labor intensive for laboratory technicians using these methods. This study presents a preliminary and promising method for the clonal propagation of oak species and provides a path for cultivar development for plants belonging to the genus Quercus. Clonal propagation of oaks has long been recognized as a major challenge to selection, breeding, and improvement of the genus Quercus L. for horticultural and forestry applications. Growing Quercus from seed has been the traditional means of oak propagation (Dirr, 1987). Because of the hybridization among Quercus species and a high degree of intraspecific variation within species, oaks can express high variability in their phenotypes (Nixon, 1997). As a result, oak nursery stock tends to be variable in its tolerance to abiotic and biotic stressors, making selection and evaluation difficult. The grafting of oaks is reported as a viable method to produce asexual clones for a limited number of species (Dirr, 1987). For example, the English Oak (Quercus robur L.) has at least 262 named cultivars, many of which have been propagated by grafting and have the capacity to grow into the mature life phase (International Oak Society, 2018). Propagation using grafting methods has had limited success with a wider variety of species primarily because of the occurrence of delayed graft incompatibility (Coggeshall, 1996; Dirr, 1987; Santamour and Coggeshall, 1996). Cornell’s UHI has successfully developed a rooting protocol that uses a modified stool-bed method offering an alternative means of clonal oak propagation (Amissah and Bassuk, 2009; Griffin and Bassuk, 1996). The breeding and development of oak cultivars has been a long-term goal for the UHI. The genus Quercus is a species-rich clade of long-lived trees that inhabit a wide variety of ecological conditions across their natural range and provide a rich genetic source for plant breeding material (Cavender-Bares, 2019). Oaks are generally known to be drought tolerant, making them ideal candidates for planting in typically low soil moisture conditions found in the urban environment (Abrams, 1990; Osuna et al., 2015; Sj€oman et al., 2018). Oaks are commonly a major component of urban forests across eastern North American cities and are frequently requested by residents as a desirable street tree (Raupp et al., 2006). Despite the popularity of oaks as street trees, their tolerance to urban site conditions is often mixed due to the genetic variability of seed sources from which the trees are grown. Development of oak cultivars would allow for assessing specific genotypes for their tolerance to urban stressors and lead to increased health and performance of oaks in the urban forest. The development of a modified stool-bed method of clonal oak propagation has removed a key technical barrier to initiating an oak breeding program (Amissah and Bassuk, 2009; Griffin and Bassuk, 1996). With the propagation constraint overcome, a white oak breeding programwas undertaken by the UHI from 2004 to 2006 and resulted in the development of 345 unique interspecific genotypes. Crosses were made with seven maternal parent taxa and 36 paternal parent taxa from species native to North America, Europe, and Asia (Denig et al., 2013). Paternal germplasm selection focused on species that grow under natural conditions that were analogous to stressors found in the urban environment such as regions with high soil pH or frequent droughts. Hybrid oak seedlings from the breeding program were grown continuously at Cornell’s Bluegrass Lane Turf and Landscape Research Center [USDA plant hardiness zone 5b (–15 to –10 F)] in Ithaca, NY. In 2013, a variation of the stoolbed method was successfully applied to UHI hybrid white oaks resulting in a series of rooted trees that were then evaluated (Denig et al., 2013). Evaluation of UHI hybrid oaks for tolerance to the urban environment has included screening hybrid oaks for alkaline tolerance in high pH soils (Denig et al., 2014); a study of UHI hybrid oaks capacity to use the osmotic adjustment drought tolerance mechanism for trees growing under field conditions (Schwartz Sax, 2019); pest (Cynipid wasp gal, Japanese beetles); and disease (powdery mildew, anthracnose) tolerance (Schwartz Sax, 2019). Additionally, evaluation of UHI hybrid oaks was undertaken to assess tree form, leaf morphology, fall color, ornamental quality, and general performance growing under nursery and field conditions. While the modified stool-bed method proved successful for the clonal propagation of a series of hybrid white oaks, the number of individual trees produced in an annual cycle was low for most genotypes (Denig et al., 2013). For example, UHI hybrid oak genotypes annually produced an average four to eight shoots per individual stock plant. For each of these shoots, the probability of producing roots ranged from 19.4% to 60.0%, depending on genotype and maternal parentage (Denig et al., 2013). This limitation reduced the commercial viability of the stool-bed rooting method. To overcome this limitation, tissue culture protocols were trialed to determine if in vitro methods could successfully be used to clonally propagate UHI hybrid oaks. The use of oak tissue culture methods used to grow plants beyond an organoid phase Received for publication 27 June 2019. Accepted for publication 22 Sept. 2019. Current address for M.S.S. : Arboretum Director, Adjunct Associate Professor of Botany, Connecticut College, 103 Olin Science Center, New Lon-