Buddleja L., commonly known as butterfly bush, encompasses ≈125 species distributed throughout most of the world, including North and South America, Asia, and Africa (Norman, 2000). The majority of the species are either diploid (2n = 38) or tetraploid (2n = 76), although 16-ploid (2n = 304) species have been reported (Moore, 1960). Within the tetraploid species, B. davidii Franch. and its numerous cultivars are the most important economically, because they are commonly found throughout the nursery trade. Breeders are trying to incorporate useful traits found in other Buddleja species back into B. davidii; however, chromosome number incompatibilities and sterility problems can limit success rate.
Such was the case in 2001, when an original cross made by Raymond Moore in 1949 (reported by Leeuwenberg, 1979) between diploid B. madagascarensis Lam. and the diploid B. crispa Benth was repeated (Renfro, 2004). Hybrids with multiple panicles of dense orange flowers that retained the nondehiscent fruit characteristic resulted from this intersectional cross between B. madagascarensis (section Nicodemia) and B. crispa (section Buddleja). Resulting hybrids were sterile; therefore, incorporating its unique traits into B. davidii would be difficult. Similar sterility problems were also reported in F1 progeny after intersectional hybridization of diploids B. madagascarensis × B. asiatica Lour. (Tobutt and Prevette, 1993). Not all intersectional crosses result in sterile progeny, as Lindstrom et al. (2004) reported fertile progeny resulting from the intersectional cross B. indica Lam. (section Nicodemia) by B. davidii ‘White Bouquet’ (section Buddleja).
Recovering fertility and equalizing chromosome numbers by mitotic polyploidization can be achieved by using mitotic inhibitors. The most commonly used chemical mutagens in horticulture are colchicine and oryzalin. Using colchicine, Rose et al. (2000) produced a tetraploid B. globosa Hope. Colchicine has been used for many years, although oryzalin is proving to be an effective substitute because it was found to be as effective as colchicine at significantly lower concentrations and is less hazardous to human health (Kermani et al., 2003; Morejohn et al., 1987). Both act as spindle inhibitors by disrupting spindle microtubules in mitosis; however, colchicine has been shown to cause mutagenic effects as well (Luckett, 1989; Tambong et al., 1998). Success rate using these chemicals for chromosome doubling varies depending on the plant species, concentration levels, and treatment duration. Restored fertility, increased flower and leaf sizes, and shortened internodes often accompany ploidy manipulation (Pryor and Frazier, 1968).
Plants treated with chemical inhibitors must be screened to identify polyploid plants. Flow cytometry is recognized as a quick way to confirm and sort plants based on relative DNA content of nuclei without having to do labor-intensive and often subjective stomatal or chromosome root-tips counts (Galbraith et al., 1997).
The goal of this research was to determine by flow cytometry if oryzalin could be used to double the chromosomes of an orange-flowered diploid interspecific hybrid (B. madagascarensis × B. crispa) as described by Renfro (2004). The overall objective was to restore the fertility of the sterile hybrid so that novel traits could be introgressed into the cultivated gene pool of B. davidii.
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