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; Sjöman 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 program was 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 stool-bed 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 was first reported 1985, and the methods have continued to be refined and improved (Vieitez et al., 1985, 2012). Contemporary oak tissue culture methods primarily include shoot cultures and somatic embryogenesis (Vieitez et al., 2012). Shoot culture methods have been used to successfully grow taxa of conservation concern (Brennan et al., 2017; Kramer and Pence, 2012) and economically important North American species in the white oak sections (sect. Quercus) and red oak sections (sect. Lobatae) (Vieitez et al., 2009). Although shoot culture methods have successfully been applied to a series of oak species, specific challenges exist in the tissue culture process. These challenges have included relatively few genotypes within a given species being responsive to the tissue culture environment (genotype specificity) (Herrmann and Buscot, 2008; San-José et al., 1988; Vengadesan and Pijut, 2009; Vieitez et al., 1993, 1994, 2009); terminal shoot tips dying after prolonged periods of growing in tissue culture medium (shoot tip necrosis) (Schwarz and Schlarbaum, 1993; Vieitez et al., 1994, 2009); and episodic growth patterns of oaks in tissue culture (Herrmann and Buscot, 2008; Vieitez et al., 2009). While these challenges have limited the widespread adoption of using tissue culture protocols for oak propagation, these methods continue to be researched and improved (Vieitez et al., 2012).
The primary objective of this study was to determine the suitability of using tissue culture methods to clonally propagate UHI hybrid oaks. To overcome specific challenges in oak tissue culture, a series of experiments were designed to improve the process. Specific objectives of this study included trialing a variety of hybrid oak germplasm for their capacity to successfully grow in the tissue culture environment and streamlining the tissue culture protocols for technicians and practitioners. The long-term goal of the UHI breeding program is to release tissue-culture propagated, stress tolerant UHI hybrid oak cultivars to the nursery and arboriculture industries.
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