Plant Cell, Tissue and Organ Culture 62: 95–100, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Plant regeneration via embryo and shoot bud formation from flower-stalk explants of Oncidium Sweet Sugar Jen Tsung Chen & Wei Chin Chang∗
Institute of Botany, Academia Sinica, Taipei, Taiwan 115, Republic of China (∗ requests for offprints; Fax: 886-22782-7954; E-mail:
[email protected]) Received 25 November 1999; accepted in revised form 22 August 2000
Key words: callus, Oncidium, shoot bud formation, somatic embryogenesis, thidiazuron
Abstract Segments taken from flower-stalk internodes of Oncidium Sweet Sugar formed somatic embryos and shoot buds directly from wound surfaces or via nodular masses proliferation within 1.5 months, when cultured on a Gelrite-gelled 1/2-MS basal medium supplemented with thidiazuron (0.1–3 mg l−1 ) in darkness. In light, when subcultured, these nodular masses proliferated into green compact callus, and produced somatic embryos, shoot buds and/or yellowish abnormal structures spontaneously. Supplementing 0.1–1 mg l−1 NAA enhanced embryo formation, but retarded proliferation of shoot buds and yellowish abnormal structures. Somatic embryos that directly formed from wound surfaces of flower stalk explants usually developed into abnormal structures, but the callus-derived embryos could germinate into PLBs and eventually developed to normal plantlets on a hormonefree basal medium for 3–4 weeks. Both the embryo-and shoot bud-derived regenerants developed into healthly plantlets when potted in sphagnum moss and acclimatized in the greenhouse. Abbreviations: MS – Murashige and Skoog basal medium; NAA – naphthaleneacetic acid; PLB – protocorm-like body; TDZ – 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea; 2,4- D – 2,4-dichlorophenoxyacetic acid Introduction The genus Oncidium (Orchidaceae), with over 750 species, is native to Central America with a distribution that extends from south Florida through Mexico and the Caribbean to the Andes and Argentina (Teob, 1989). A wide range of attractive hybrids, varieties or cultivars in Oncidium has become economically important for use in cut-flower and pot-plant industries. We previously reported a reliable protocol for inducing direct somatic embryogenesis and subsequent plant regeneration from the cultured leaf tissues of orchid Oncidium Gower Ramsey on a defined medium supplemented with TDZ (Chen et al., 1999). Although these vegetative tissues taken from in vitro grown plantlets are able to form embryos at a high frequency, the mother plant may suffer from serious damage after the explants (such as leaves, stems or roots) have been taken. Floral tissues, including
flower-stalk tips (Fast, 1973), micro-inflorescences (Kerbauy, 1984) and flower-stalk buds (Nuraini and Shaib, 1992), have often been used as explants to obtain PLBs or shoots, and subsequently plantlets for the need of mass propagation of Oncidium. However, embryo formation from the floral tissues has not been reported in these early reports. In the present research, segments of flower-stalk internodes of two Oncidium cultivars (cv. Gower Ramsey and cv. Sweet Sugar) are used to induce in vitro morphogenesis on a 1/2-MS basal medium supplemented with TDZ. The early events of embryo and shoot bud formation from the explants, and subsequently plant regeneration are presented. Furthermore, the differences in the developmental patterns of the flower stalk-derived embryo of cv. Sweet Sugar and the two kinds of embryos that we previously found in leaf explants and vegetative tissue-derived calli of cv. Gower Ramsey (Chen et al., 1999) are discussed.
96 Materials and methods Three to four-year-old potted plants of Oncidium Gower Ramsey and Sweet Sugar that were grown in greenhouse, were used as donor plants. Explants 5 mm in length were excised from the internodes of the flower stalks (ca. 15–20 cm in length), when the flower buds were fully formed but the flower color had not yet appeared. The explants were surface-sterilized with 0.25% (w/v) mercuric chloride for 5 min followed by three rinses in sterile distilled water. Explants were placed on the surface of a 1/2-MS basal medium for the induction of direct somatic embryogenesis (Chen et al., 1999). The basal medium contained 1/2-strength macro- and micro-elements of Murashige and Skoog (Murashige and Skoog, 1962) supplemented with (mg l−1 ): myo-inositol (100), niacin (0.5), pyridoxine HCl (0.5), thiamine HCl (0.1), glycine (2.0), peptone (1000), NaH2 PO4 (170), sucrose (20,000), and Gelrite (2,200). Plant growth regulators were added prior to autoclaving as optional additives according to the experimental objectives. The pH of the media was adjusted to 5.2 with 1 N KOH or HCl prior to autoclaving for 15 min at 121 ◦ C. Explants were cultured in 20×150 mm culture tubes under a 16:8-h photoperiod at 28–36 µmol m−2 s−1 (daylight fluorescent tubes FL-30D/29, 40 W, China Electric Co., Taipei) and 26±1 ◦ C. Twenty replicates were taken for each treatment, and four explants were planted in each culture tube. Observations were made after 3–4 weeks of growth. The percentage embryo- and shoot-producing explants was determined for each trial. Differences between means were scored with Duncan’s multiple range test (Duncan, 1955). Cultures were examined and photographed with a stereozoom microscope (SZH, Olympus). Samples for scanning electron microscopy were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) for 4 h at 4 ◦ C, dehydrated through an ethanol series (Dawns, 1971), dried in a critical point dryer (HCP-2, Hitachi) and coated with gold in an ion coater (IB-2, Giko Engineering Co.). A scanning electron microscope (DSM-950, Carl Zeiss) was used for examination and photography of the specimen. Results Direct embryo and shoot bud formation and further development When 5-mm-long flower stalk internode explants of
Table 1. In vitro morphogenesis of flower-stalk explants of Oncidium Sweet Sugar TDZ (mg l−1 )
0 0.1 0.3 1 3
% explants produced somatic embryos
% explants produced shoot buds
10 b 15 b 15 b 60 a 65 a
0c 20 bc 40 ab 45 a 45 a
Data were scored after 2 months of culture, and are presented as % of explants forming somatic embryos or shoot buds. Percentage of 20 explants within columns followed by the same letters are not significantly different at p
