Somatic Embryogenesis and Plant Regeneration from Leaves of Dendranthema grandiflora

Somatic embryogenesis from leaf midrib explants of Dendranthema grandiflora Tzvelev. 'Iridon' cultured on modified Murashige and Skoog basal medium (MSB) containing 1.0 mg 2,4-D and 0.2 mg BA/liter was influenced by light and sucrose concentration. Somatic embryos formed directly from explants when cultured on medium con- taining 9% to 18% sucrose and incubated first in the dark for 28 days, followed by 10 days in light, and then returned to the dark for 14 days. Embryogenesis did not occur in continuous darkness and was drastically reduced when explants were incubated in light only. The most embryos were formed on medium containing either 12% or 15% sucrose; lower concentrations stimulated shoot and root development. Light also mediated embryogenesis from leaf explants of 'other cultivars. White-opaque or occasionally light-green cotyledon-stage somatic embryos germinated on MSB medium without growth regulators but containing 3% sucrose. Twelve of the 23 cultivars evaluated produced somatic embryos, but plants were recovered from only five. Regenerated plants were phenotypically similar to parent plants in growth habit, leaf morphology, and flower color. Chemical names used: N- (phenylmethyl)-1 H- purine-6- amine (BA); (2,4-dichlorophenoxy) acetic acid (2,4-D). Chrysanthemum (Dendranthema grandiflora Tzvelev. (An- derson, 1987)), an important floricultural crop in the Astera- ceae, is conventionally propagated vegetatively by stem cuttings. Chrysanthemums also have been successfully micropropagated by adventitious shoot formation from various tissues and callus cultures (Earle and Langhans, 1974; Hill, 1968; Iizuka et al., 1973; Khalid et al., 1989; Roest and Bokelmann, 1975). Tissue culture methods have provided a means to eliminate various pathogens (Ahmed and Andrea, 1987; Horst, 1990) and to in- duce and select desirable mutations (Broertjes et al., 1976; Dal- sou and Short, 1987; DeJong and Custers, 1986; Huitema et al., 1987; Preil et al., 1983). Somatic embryogenesis is another in vitro process used for regeneration and potentially offers a very efficient system for mass clonal production of horticultural crops (Ammirato and Styer, 1985; Lutz et al., 1984; Sharp et al., 1982). Although numerous species are reportedly capable of forming somatic embryos (Tisserat et al., 1979; Warm, 1988; Williams and Ma- heswaran, 1986), relatively few instances of somatic embyro- genesis have been documented for members of the Asteraceae and, among these, none are strictly ornamental or floricultural species. These reports are limited to chicory and endive, Ci- chorium species (Guedira et al., 1989; Heirwegh et al., 1985; Vasil and Hildebrandt, 1966; Vasil et al., 1964), sunflower, Helianthus annuus L. (Finer, 1987; Patterson and Everett, 1985), Brachycome lineariloba (DC) Druce (Gould, 1978), and hawk- weed, Pterotheca falconeri Hook. (syn. Crepis sanceta (L.) Babcock (Mehra and Mehra, 1971)). Earle and Langhans (1974) noted that "embryoids" were formed in callus cultures of Chry- santhemum morifolium Ramat. (D. grandiflora), but did not present substantiating evidence. Production of somatic embryos from leaf tissue and subse-