Interspecific hybridisation within Buxus spp.
Introduction
Buxus is the largest genus within the Buxaceae, a small family of evergreen trees, shrubs and rhizomatous herbs (Jarvis, 1989, Köhler and Brückner, 1989). Buxus spp., commonly known as boxwood or box plants, are broad-leaved evergreen shrubs. Their varying morphological characteristics make them popular for a wide range of ornamental uses: as individual specimens, hedges, parterres, groups, in container or as topiary plants (Larson, 1999, Batdorf, 1997, Batdorf, 2004, Van Trier and Hermans, 2005). The genus Buxus grows in a wide ecological range from dry scrub forests to limestone cliffs to the understory of montane rain and cloud forests, and can even be found above 3000 m elevation. Buxus species are distributed across five major geographical regions: (1) Europe, Mediterranean Basin and Middle East, (2) China, Japan, Korea, Malaysia and the Philippines, (3) Africa, (4) Caribbean Islands, Mexico and South America, (5) India, the northwest Himalayas and the former Soviet Union. The only areas without indigenous species of Buxus are temperate North America and Australia (Larson, 1996). European gardens commonly use cultivars of Buxus sempervirens L. as ornamental plants for hedges and topiaries. East Asian species such as Buxus microphylla Sieb & Zucc, which can stand European climatic conditions, are also planted on occasion. Within Buxus microphylla, four groups are distinguished: the koreana group, the sinica group, the japonica group and the microphylla group (Van Trier and Hermans, 2005). Other species growing in temperate regions are Buxus balearica Lam., Buxus bodinieri Léveillé, Buxus colchica Pojark, Buxus harlandii Hance, Buxus henryi Mayr. and Buxus wallichiana Ball. (Larson, 1999). Phylogenetic studies based on the development and structure of inflorescences and flowers (von Balthazar and Endress, 2002a, von Balthazar and Endress, 2002b) and pollen characteristics (Köhler, 1994) have been made. Genetic relationships in Buxaceae have also been described (von Balthazar et al., 2000, Van Laere et al., 2011). These studies reveal a great deal of morphological, genetic and cytogenetic variation among the Buxus species.
As is typical for most woody plants, breeding efforts within Buxaceae have been limited. The main reasons are the long life cycles and the high cost of breeding. Important sources of novelties within Buxus are selections from ‘wild’ populations, ‘lucky finds’ in open-pollinated seedling populations and spontaneously occurring mutations (Van Huylenbroeck and Van Laere, 2010). Controlled pollinations have not yet been fully exploited within Buxus, although the present morphological and genetic variation within Buxus species presents opportunities to develop novel cultivars through interspecific hybridisation (Van Laere et al., 2011). To our knowledge, within Buxus, only interspecific hybridisation between Buxus sempervirens and B. microphylla var. koreana has been reported (Van Trier and Hermans, 2005), resulting in hybrids such as Buxus ‘Green Mound’, Buxus ‘Green Velvet’, Buxus ‘Green Gem’ and Buxus ‘Green Mountain’. AFLP analysis confirmed the hybrid character of these cultivars (Van Laere et al., 2011). Interspecific hybrids have the potential to capture hybrid vigour as well as to combine traits that do not occur within a single species. Many techniques already exist to overcome pre- and postzygotic barriers (Van Tuyl and Lim, 2003, Eeckhaut et al., 2006). Interspecific hybrids can alter not only the qualitative traits but also the quantitative traits of the parent species. Besides morphological traits, such as growth habit, leaf colour, etc., also physiological features, disease resistance, winter hardiness and leaf discoloration are very important for Buxus. The most important worldwide threat for boxwood today is the disease caused by Cylindrocladium buxicola disease (commonly known as box blight), which causes dark brown spots on the leaves, black streaks on the stems and severe defoliation. This fungal disease can spread rapidly throughout the plant under warm and humid conditions (Henricot et al., 2000, Henricot et al., 2008, Henricot and Culham, 2002, Gehesquière et al., 2013). Differences in disease resistance are seen between species (Henricot et al., 2008, Gehesquière, 2014), which opens the possibility to breed for more disease resistance via interspecific hybridisation.
As little information is available about interspecific hybridisation and the occurrence of pre- and postzygotic barriers in Buxus, the aim of present study was to explore the possibilities for interspecific hybridisation within Buxus. We have performed interspecific and interploidy crosses. The hybrid character of the resulting F1 seedlings was verified using molecular markers and flow cytometry.
Section snippets
Plant material
For B. sempervirens, a wild genotype and the cultivars ‘Latifolia Maculata’, ‘Rotundifolia’, ‘Angustifolia’, ‘Hermans Low’, ‘Suffruticosa’, ‘Handsworthiensis’, ‘Dark Sky’ and ‘Glauca’ were used. Within B. microphylla, var. koreana from the koreana group, ‘Faulkner’, ‘National’ and ‘Sunnyside’ from the sinica group, ‘Trompenburg’ from the japonica group and ‘Rococo’ from the microphylla group were used. Other species used were B. harlandii, B. henryi, B. balearica and B. colchica. The plants
Interspecific hybridisation and seed germination
In total, 7649 interspecific pollinations yielded 14,928 seeds and 4750 seedlings were obtained. As a control 1625 intraspecific pollinations were performed within B. sempervirens and B. microphylla. These crosses resulted in 2131 seeds and 318 seedlings (Table 2).
On average 1.31 (for intraspecific) and 1.95 (for interspecific) seeds were obtained per pollination. Pollinations involving B. harlandii were most successful with more than 4 seeds per pollination. Only the cross B. microphylla × B.
Discussion
In Buxus, interspecific hybridisation was not hindered by prezygotic barriers since pollen tube growth through the style towards the ovaria could be observed (data not shown). For most cross combinations fruits and seeds were obtained and the resulting seeds matured fully on the plants. This is in contrast with observations in many other genera in which an in vitro embryo rescue step is required to gain interspecific seedlings after distant crosses (Sharma, 1995, Burke and Arnold, 2001, Jansky,
Conclusion
To our knowledge, this study is the first to provide an in-depth exploration of the possibilities of interspecific hybridisation in Buxus. To date, only interspecific crosses between B. sempervirens and B. microphylla var. koreana are reported (Van Trier and Hermans, 2005). We have demonstrated that within Buxaceae no prezygotic barriers exist. However, postzygotic barriers, mainly lack of germination of the seeds and unilateral incongruity, do occur. Existing ploidy differences in Buxus
Acknowledgements
The authors are grateful to Frederik Delbeke and Jorien Oomen for technical assistance in the greenhouse and in the lab. The authors also want to thank Jan De Riek for his assistance in analysing the AFLP results.
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