In Vitro Culture of Zygotic Embryos of Taxus Species

An embryo culture method overcomes the lengthy dormancy requirement of Taxus L. spp. (yew) seeds. When zygotic embryos excised from mature T. brevifolia L. seeds were cultured in darkness for 4 weeks on one of three basal salt media (B5, Litvay, and Murashige and Skoog), radicle emergence and seedling development was highest on B5 basal salt medium. After 1 to 2 weeks on B5 basal salt medium, seedling development of T. brevifolia, T. cuspidata L., T. baccata L., and T. baccata stricta L. ranged from 2% to 36%. BA at 2.25 μM had no effect on radicle emergence; 22.5 μM prevented it. Embryos excised from mature or nearly mature seeds had the highest frequency of radicle emergence and seedling development. Cultured embryos developed seedlings in only 8 to 10 weeks. Chemical name used: N -benzyladenine (BA). Taxus brevifolia is a slow-growing evergreen species occurring primarily in ancient forests of the Pacific Northwest in the United States. Interest in the culture of this and other Taxus species has intensified with the discovery of taxol, an anticancer drug found in various parts of the tree (Vidensek et al., 1990; Witherup et al., 1990). The total synthesis of taxol has recently been reported (Nicolaou et al., 1994); however, because of the complexity and numerous steps involved, large-scale synthesis is not now feasible. Because T. brevifolia grows slowly and has a lengthy seed dormancy requirement ( 1.5 to 2 years), the supply of taxol is limited (Steinfeld, 1992). Embryo culture may overcome the problems posed by lengthy dormancy (Collins and Grosser, 1984). Flores and Sgrignoli (1991) reported an embryo culture method for Taxus that could overcome the dormancy requirement. However, their method was based on experiments that included few excised zygotic embryos. Therefore, the objective of the present research was to evaluate the response of excised T. brevifolia embryos to three basal salt culture media, i.e., B5 (Gamborg et al., 1968), LV (Litvay et al., 1985), and MS (Murashige and Skoog, 1962). Two additional objectives were to determine embryo responses of T. brevifolia, T. cuspidata, T. baccata, and T. baccata stricta in B5 culture medium, and whether seed maturity influences the frequency of radicle emergence and seedling development in Taxus. Materials and Methods Plant material and embryo isolation. Mature T. baccata, T. cuspidata, and T. baccata Receivedforpublication12Oct. 1993. Accepted for publication 18 Mar. 1994. I thank Thomas Vilmar forassistance regarding statistical analysis of data. The cost of publishing this paper was defrayed in part by the payment of page charges, Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. HORTSCIENCE, VOL. 29(6), JUNE 1994 stricta seeds were supplied by F.W. Schumacher Co., Sandwich, Mass. Taxus brevifolia seeds were provided by Special Trees, Portland, Ore. Seeds were surface-sterilized for 10 min in concentrated HC1, and then rinsed five times in sterile, distilled water. After sterilization, seeds were soaked in sterile, distilled water for 24 h. Embryos were excised aseptically from surrounding gametophytic tissue under a binocular microscope using fine forceps and a scalpel. Embryo culture. Zygotic embryos were cultured on B5, LV, or MS basal medium. All basal salt media were supplemented with 3% sucrose and solidified with 0.7% Phytagar (Gibco-BRL, Gaithersburg, Md.). In experiments where the effects of a growth regulator on embryo germination were studied, embryos were cultured on B5 basal medium supplemented with BA at 0, 2.25, or 22.5 μM. The pH of all media was adjusted to 5.8 before autoclaving at 121C for 20 min. Generally, 20 embryos were placed in a 100 × 20-mm petri dish containing 25 ml of culture medium. In cases where 20 embryos were not obtainable, some replicate dishes contained either more or fewer embryos. After transferring embryos to nutrient media, petri dishes were sealed with parafilm and incubated for 4 weeks at 26C in darkness. Subsequently, cultures were transfer-red to fresh medium of the same formulation and maintained under a 16-h photoperiod provided by cool-white fluorescent lamps (80 μmol·m2·s-1). Seedlings were transferred to soil after they developed leaves and branched root systems. Developmental stages of seeds. Seed maturity was classified into three stages according to the color of the seeds and status of fleshy arils. Stage I (young seeds): seeds were ≈2 mm long and light to medium green; the pre-aril sheath of young seeds was unswollen and green. Stage II (intermediate seeds): seeds were ≈4 mm long and dark green or light brown; the pre-aril sheath had begun to swell and was light pink. Stage III (mature seeds): seeds were ≈6 mm long and brown; the aril was swollen and red. Statistical analyses. Germination or breaking dormancy was defined as radicle emergence, accompanied by embryo greening and elongation. Percent seedling development was defined as the number of embryos that ultimately developed into seedlings. The total number of embryos cultured in each treatment of each experiment is reported in Tables 1–5. For each treatment, data were entered into a contingency table. The response or nonresponse of different treatment groups was compared using the statistical procedure CATMOD (SAS Institute, 1989). Results and Discussion Formulation of basal salt. Radicle emergence frequency of T. brevifolia embryos cultured on B5, LV, and MS basal salts media was between 50% and 60% (differences nonsignificant), but final seedling development was lowest with LV, with MS being intermediate (Table 1). The effectiveness of the tested media on plant development was ranked as B5 > MS > LV. Light vs. darkness. Radicle emergence of excised T. baccata stricta embryos was not affected by light; T. baccata radicle emergence was superior in darkness (Table 2). Darkness effected a greater response on seedling development for both species. Overall, T.