Summary
Leaf segments of the orchid sp. Phalaenopsis ‘Little Steve’ were used as explants testing the effects of 2,4-dichlorophenoxyacetic acid (2,4-D; 0.45, 2.26, 4.52 μM), 6-furfurylaminopurine (kinetin; 2.32, 4.65, 13.95 μM), N6-benzyladenine (BA; 2.22, 4.44, 13.32 μM), and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 2.27, 4.54, 13.62μM) on the induction of direct somatic embryogenesis. After 20–30 d of culture in darkness, clusters of somatic embryos formed from leaf surfaces and wounded regions of explants on half-strength Murashige and Skoog medium supplemented with BA and TDZ. However, kinetin had no response on direct embryo induction. In addition, 2,4-D highly retarded the frequency of embryogenesis that was induced by TDZ. Generally, adaxial surfaces near wounded regions had the highest embryogenic competency compared to other regions of explants. Histological sections revealed that somatic embryos mostly arose from epidermal cell layers of the explants. Secondary embryogenesis occurred at basal parts of embryos, and originated from outer cell layers. Following transfer of regenerated embryos onto growth regulator-free medium for 3.5–4 mo., plantlets with three to four leaves and several roots were obtained. This protocol provides a simple way to regenerate plants through direct somatic embryogenesis, and is suitable for further studies on embryo development and genetic transformation of Phalaenopsis.
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References
Arditti, J.; Ernst, R. Micropropagation of orchids, New York: John Wiley & Son; 1993:467–520.
Chen, J. T.; Chang, C.; Chang, W. C. Direct somatic embryogenesis from leaf explants of Oncidium ‘Gower Ramsey’ and subsequent plant regeneration. Plant Cell Rep. 19:143–149; 1999.
Chen, J. T.; Chang, W. C. Efficient plant regeneration through somatic embryogenesis from callus cultures of Oncidium (Orchidaceae). Plant Sci. 160:87–93; 2000.
Chen, J. T.; Chang, W. C. Effects of auxins and cytokinins on direct somatic embryogenesis from leaf explants of Oncidium ‘Gower Ramsey’. Plant Growth Regul. 34:229–232; 2001.
Chen, J. T.; Chang, W. C. Induction of repetitive embryogenesis from seed-derived protocorms of Phalaenopsis amabilis var. formosa Shimadzu. In Vitro Cell. Dev. Biol. Plant 40:290–293; 2004.
Chen, Y.; Piluek, C. Effects of thidiazuron and N6-benzylaminopurin on shoot regeneration of Phalaenopsis. Plant Growth Regul. 16:99–101; 1995.
Chen, Y. C.; Chang, C.; Chang, W. C. A reliable protocol for plant regeneration from callus culture of Phalaenopsis. In Vitro Cell. Dev. Biol. Plant 36:420–423; 2000.
Duan, J. X.; Chen, H.; Yazawa, S. In vitro propagation of Phalaenopsis via culture of cytokinins-induced nodes. J. Plant Growth Regul. 15:133–137; 1996.
Duncan, D. B. Multiple range and multiple F test. Biometrics 11:1–42; 1955.
Ernst, R. Effects of thidiazuron on in vitro propagation of Phalaenopsis and Doritaenopsis (Orchidaceae). Plant Cell Tiss. Organ Cult. 39:273–275; 1994.
Ishii, Y.; Takamura, T.; Goi, M.; Tanaka, M. Callus induction and somatic embryogenesis of Phalaenopsis. Plant Cell Rep. 17:446–450; 1998.
Islam, M. O.; Ichihashi, S. Effects of sucrose, maltose and sorbitol on callus growth of Phalaenopsis, Doritaenopsis and Neofinetia. J. Jap. Soc. Hort. Sci. 68:1124–1131; 1999.
Jensen, W. A. Botanical histochemistry. San Francisco: Freeman; 1962.
Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.
Park, S. Y.; Murthy, H. N.; Pack, K. Y. Rapid propagation of Phalaenopsis from floral stalk-derived leaves. In Vitro Cell. Dev. Biol. Plant 38:168–172; 2002.
Tanaka, M.; Hasegawa, A.; Goi, M. Studies on the clonal propagation of monopodial orchids by tissue culture. I. Formation of protocorm-like bodies from leaf tissues in Phalaenopsis and Vanda. J. Jap. Soc. Hort. Sci. 44:47–58; 1975.
Teob, E. S. Orchids of Asia. Singapore: Times Books International; 1989:125–134.
Tokuhara, K.; Mii, M. Micropropagation of Phalaenopsis and Doritaenopsis by culturing shoot tips of flower stalk buds. Plant Cell Rep. 13:7–11; 1993.
Tokuhara, K.; Mii, M. Induction of embryogenic callus and cell suspension culture from shoot tips excised from flower stalk buds of Phalaenopsis (Orchidaceae). In Vitro Cell. Dev. Biol. Plant 37:457–461; 2001.
Young, P. S.; Murthy, H. N.; Yoeup, P. K. Mass multiplication of protocorm-like bodies using bioreactor system and subsequent plant regeneration in Phalaenopsis. Plant Cell Tiss. Organ Cult. 63:67–72; 2000.
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Kuo, HL., Chen, JT. & Chang, WC. Efficient plant regeneration through direct somatic embryogenesis from leaf explants of Phalaenopsis ‘Little Steve’. In Vitro Cell.Dev.Biol.-Plant 41, 453–456 (2005). https://doi.org/10.1079/IVP2005644
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DOI: https://doi.org/10.1079/IVP2005644