H ORTS CIENCE 26(10):1322-1324. 1991. Histology and Morphology of Asparagus Somatic Embryos A. Levi l and K.C. Sink Department of Horticulture, Michigan State University, East Lansing, MI 48824 Additional index words. Asparagus officinalis, embryogenesis, tissue culture Abstract. The histology and morphology of developing asparagus Asparagus officin- alis L.) somatic embryos arising in callus cultures were examined and contrasted with that documented for zygotic embryos. Histological sections of lateral bud-derived callus cultured for 2 weeks on embryo induction medium consisting of Murashige and Skoog salts and vitamins (MS) with 1.5 mg NAA/liter and 0.1 mg kinetin/liter indicated the formation of distinct groups of embryogenic cells. At 4 weeks, the callus was comprised of embryos in the early and late globular stages and a few bipolar embryos. Within 2 weeks on embryo development medium consisting of MS with 0.05 mg NAA/liter and 0.1 mg kinetin/liter, the globular embryos developed a bipolar shape having an ex- panded upper region that formed the cotyledon and a smaller region that formed the radicle. Within 4 to 6 weeks on this latter medium, each mature bipolar embryo was opaque and had a large cotyledon, a distinct shoot apex at the cotyledon-hypocotyl junction, and vascular connections between the radicle, shoot apex, and cotyledon. Many mature somatic embryos resembled the asparagus zygotic embryos in having a crescent shape, whereas others had a short but wide cotyledon. Both somatic embryo types converted to plantlets at equal rates. Chemical names used: N- (2-furanylmethyl)- 1 H -purin-6-amine (kinetin); 1-naphthaleneacetic acid (NAA). Asparagus has a low multiplication rate using conventional propagation methods (Yang and Clore, 1973). Tissue culture ex- periments have proved to be successful, and asparagus is one of the first monocot species to be regenerated to somatic embryos and plantlets in vitro (Reuther, 1977; Steward and Mapes, 1971; Wilmar and Hellendoorn, 1968). However, the developmental se- quence of in vitro-derived somatic embryos has not been histologically detailed nor con- trasted to that of asparagus zygotic embryos (Riviere, 1973). To date, only the gross morphology of the crescent-shaped somatic embryo stage (Bui Dang Ha et al., 1975) and a schematic pattern of the multicellular ori- gin of embryos (Reuther, 1983) have been reported. Recently, we demonstrated that calli derived from lateral buds and crowns in vitro had a higher embryogenic capacity than calli derived from spear sections (Levi and Sink, 1991). Additionally, NAA promoted a higher frequency of normal somatic embryos than did 2,4-dichlorophenoxyacetic acid (2,4-D). This report examines the developmental se- quence of asparagus somatic embryos formed Received for publication 2 Jan. 1991. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regula- tions, this paper therefore must be hereby marked advertisement solely to indicate this fact. 1 Present address: Dept. of Horticulture, Univ. of Georgia, Athens, GA 30602. in lateral bud-derived calli and contrasts it with that already known for asparagus zy- gotic embryos. Lateral buds (1 to 2.5 cm long) were ob- tained from the upper portion of 4- to 7-day- old spears of a male asparagus crown selec- tion (M.S.U. C-3) grown in the greenhouse. Spears were surface sterilized for 30 min in aqueous sodium hypochlorite (1.5% w/v) so- lution with 0.05% Tween 20. Subsequently, spears were rinsed three times with sterile distilled water. To initiate callus, three lateral buds were placed in each of ten 15 × 100-mm petri dishes. Each petri dish contained 25 ml of culture medium: Murashige and Skoog (1962) (MS) with 0.1 mg NAA/liter and 0.01 mg kinetin/liter. Within 4 weeks, primary calli were separated from the explants and sub- culture twice at 4-week intervals. Subse- quently, calli were transferred to MS medium with 1.5 mg NAA/liter and 0.1 mg kinetin/ liter (embryo induction medium). Following 4 weeks on induction medium, calli were transferred to MS medium with 0.05 mg NAA/liter and 0.1 mg kinetin/liter (embryo development medium). Embryos that con- verted to plantlets were rooted on MS me- dium with 2% glucose. All culture media, except as noted, con- tained 3% sucrose, were solidified with 0.9% Bacto agar, and were adjusted to pH 5.9 be- fore autoclaving. Callus initiation and em- bryo induction cultures were incubated in the 1322 HORTSCIENCE , VOL. 26(10), OCTOBER 1991