Somatic Embryogenesis and Plant Regeneration in Cucumber

The embryogenic capacity of seven cucumber (Cucumis sativus L.) cultivars was examined by tissue culture of cotyledon, young first-leaf, and internode explants. Somatic embryogenesis frequencies differed significantly among the tested cultivars, and ‘Fushinarimidori’ produced the highest number of embryos from either cotyledons or young first leaves. Cotyledonand first-leaf-derived calluses produced more embryos than calluses from internodes. Somatic embryos were induced from ‘Aonaga F1’ internodes. With relatively high sucrose levels (6% and 9%) in the initiation medium, the frequency of embryogenic callus formation from ‘Fushinarimidori’ cotyledon explants was >90%. The highest yield of somatic embryos occurred in cultures initiated with high sucrose levels (9% or 12%), although 12% sucrose inhibited callus formation and growth. Somatic embryos germinated in a basal liquid medium supplemented with 0.5% activated charcoal, and they developed into well-shaped, healthy plantlets on semisolid medium with 1% sucrose. Since Malepszy et al. (1982) reported the formation of somatic embryos in cucumber, researchers have made remarkable progress in somatic embryogenesis. Somatic embryos have been induced from various explants, such as cotyledons, hypocotyls, true leaves, or other explants (Bergervoet et al., 1989; Cade et al., 1988, 1990; Chee, 1990; Chee and Tricoli, 1988; Malepsey and Nadolska-Orczyk, 1983; Rajasekaran et al., 1983; Tabei and Kanno, 1989; Trulson and Shahin, 1986; Ziv and Gadasi, 1986). To our knowledge, however, there is no report on successful plantlet regeneration from internode explants via somatic embryogenesis in cucumber. In general, a low frequency of somatic embryogenesis is common in published reports. Moreover, a relatively high frequency of abnormal embryogenesis, such as disordered bipolarity, embryo death, or arrest of growth in developmental stage, recallus, and vitrification of embryos has been reported for cucumber (Bergervoet et al., 1989; Cade et al., 1988, 1990; Jia et al., 1988; Kim and Janick, 1989; Ladyman and Girard, 1992; Ziv and Gadasi, 1986). In out study, we investigate the influence of cultivar, explant source, and sucrose concentration on embryogenic callus formation and somatic embryogenesis. Materials and Methods Seven cucumber cultivars were used in Expt. 1 (‘Aonaga F1’, ‘Aonagajibai’, ‘Chikanarishiyou’, ‘Fushinarimidori’, ‘Hokushin’, 906 Received for publication 20 Sept. 1993. Accepted for publication 28 Jan. 1994. 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. ‘Shimoshirazu’, and ‘Yoshinari’); only ‘Fushinarimidori’ was used in Expt. 2. Seeds were surface-disinfested for 15 min in 1.0% sodium hypochlorite solution with 3 drops Tween 20/100 ml, followed by three rinses in sterile distilled water. For germination, seeds were placed on basal Murashige and Skoog (1962) medium with 3% (w/v) sucrose, 0.25% (w/v) Gelrite (San-Ei Co., Osaka, Japan), and Gamborg’s B5 medium vitamins (Gamborg et al., 1968). All media pH was adjusted to 5.8 with 0.5 N KOH before adding Gelrite and autoclaving for 15 min at 121C. Unless otherwise stated, cultures were maintained under a 16-h photoperiod at 29 μmol•m•s photosynthetic photon flux (PPF) provided by cool-white fluorescent lamps, at 25 ± 1C (80% ± 5% relative humidity). Cultivars and explant sources (Expt. 1). Cotyledon, first-leaf, and internode explants from 4-week-old seedlings were excised with a scalpel; cotyledons and first leaves were cut into 10 or 12 disks (4 to 6 mm), and the internodes were cut into 3to 4-mm-long sections. All explants were excised on the same day and placed on initiation medium [the basal medium supplemented with 9.0 μM 2,4dichlorophenoxy acetic acid (2,4-D), 2.7 μM 1-naphthaleneacetic acid (NAA), and 2.2 μM 6-benzylaminopurine (BA)] for 3 weeks. Two replications were used in this experiment, and for each replication, 24 explants were cultured. Calluses were transferred onto embryo induction medium [the basal medium containing 2.7 μM NAA, 2.3 μM kinetin, and 6% (w/v) sucrose] for an additional 2 to 3 weeks of culture. We determined the ability of explants to undergo somatic embryogenesis by counting the number of embryos per explant and the number of calluses forming embryos. Means were compared using Duncan’s multiple range test. The interacting effects of cultivars and explant source on somatic embryogenesis were determined using analysis of variance. Sucrose concentrations (Expt. 2). Cotyledon explants derived from 6to 7-day-old seedlings of ‘Fushinarimidori’ (which produced the most somatic embryos in Expt. 1) were cultured on the initiation medium containing 3%, 6%, 9%, or 12% (w/v) sucrose. After 3 weeks, the proliferating explants with or without calluses were transferred to the embryo induction medium supplemented with 2.7 μM NAA; 2.3 μM kinetin; and containing 3%, 6%, or 9% (w/v) sucrose; or supplemented with 3% sucrose and no plant growth regulators (PGR). The percentage of explants that formed somatic embryos and the number of embryos formed were recorded 5 to 6 weeks after explants were first placed in vitro. Means were compared and evaluated using Duncan’s multiple range test or least significant difference. The interacting effects between sucrose levels in the initiation medium and the embryo induction medium on the formation of somatic embryos were determined using analysis of variance. Development into plantlets. Somatic embryos in the cotyledonary stage with or without visible radicles obtained from the embryo induction medium (after 2 to 3 weeks of culture) were cultured into 200-ml Erlenmeyer flasks containing 15 ml of liquid modified basal medium, supplemented with 0.5% (w/v) activated charcoal (Wako Pure Chemical Industries Co., Osaka, Japan), 1.5% (w/v) sucrose, and 1.5% (w/v) glucose but no PGR. After 1 or 2 weeks of rotatory culture at 1 rpm, the cotyledonary-stage embryos developed cotyledons and roots and grew into whole plants; then they were transferred onto the semisolid basal medium containing 1.0% (w/v) sucrose and held under a 47-μmol•m•s PPF with a 16-h photoperiod for 1 week. Plantlets with well-developed cotyledons and roots were transplanted to φ80 × 100-mm plastic boxes containing autoclaved fine vermiculite wetted with 0.1% (w/v) 6.5N–6P– 19K Hyponex (Hyponex Co., Marysville). To harden plantlets, the caps of the containers were opened gradually, until they were completely removed. Hardened cucumber plants were transferred to pots under greenhouse conditions, where they flowered and produced fruit.