Efficient plant regeneration through direct somatic embryogenesis from leaf explants of Phalaenopsis ‘Little Steve’

Abstract

SummaryLeaf 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 mM), 6-furfurylaminopurine (kinetin; 2.32, 4.65, 13.95 mM), N 6 -benzyladenine (BA; 2.22, 4.44, 13.32 mM), and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 2.27, 4.54, 13.62 mM) 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.

Role of Auxin in Orchid Development

“…62,63 Researchers have also demonstrated that auxin can inhibit cytokinin-induced direct somatic embryogenesis in Phalaenopsis and Oncidium species. 64,65 Overall, these findings suggest that high exogenous auxin levels promote and/or maintain undifferentiated growth, while hormonedepleted media or lower levels of auxin in conjunction with cytokinins typically produce PLBs from callus. This conclusion further supports the premise that, in vivo, uniform auxin distribution helps to maintain a dedifferentiated protocorm until auxin is redirected to appropriate locations and is present in precise concentrations to help stimulate differentiation (Fig.…”

Embryogenic Callus Induction from Leaf Tip Explants and Protocorm-Like Body Formation and Shoot Proliferation of Dimorphorchis lowii: Borneon Endemic Orchid

“…TDZ is a substituted phenyl urea with cytokinin-like activity (van Staden, Zažímalová, & George, 2008;Jitsopakul, Thammasiri, & Ishikawa, 2013). The ascendancy of TDZ in inducing somatic embryos (infant PLBs) from leaf explants was recounted in numerous orchid species including Oncidium (Mayer, Stancato, & Appezzato-Da-Glória, 2010), Doritaenopsis (Park, Yeung, Chakrabarty, & Paek, 2002), Dendrobium and Phalaenopsis (Kuo, Chen, & Chang, 2005). TDZ is also useful for rapid plant regeneration through organogenesis (Kishor & Devi, 2009;Jitsopakul, Thammasiri, & Ishikawa, 2013), somatic em-bryogenesis (Jheng, Do, Liauh, Chung, & Huang, 2006;Gow, Chen, & Chang, 2009), or PLB formation of orchids (Mayer, Stancato, & Appezzato-Da-Glória, 2010;Khoddamzadeh et al, 2011;Roy, Sajeev, Pattanayak, & Deka, 2012).…”

“…Most of the time, callus developed at the cut ends of leaf tip explants, suggesting that the wounding might have triggered dedifferentiation of cells to form callus. Kuo, Chen, & Chang (2005) reported that the highest ability of Phalaenopsis leaf explants to form embryos was found at adaxial surfaces near cut ends on TDZ-containing medium. Figure 3C-D show that the callus masses composed of spherical, compact and loose aggregates of cells.…”

“…Among them are Aerides (Devi, Devi, & Singh, 2013), Aranda (Gantait, Bustam, & Sinniah, 2012), Phalaenopsis (Kuo, Chen, & Chang, 2005;Khoddamzadeh et al, 2011), Vanda and Dendrobium (Martin & Madassery, 2006;Tee, Wong, Lam, & Maziah, 2010), and Oncidium (Mayer, Stancato, & Appezzato-Da-Glória, 2010). In leaf cultures, for example in Renanthera (Wu et al, 2012) and Vanilla (Janarthanam & Seshadri, 2008), wound surface of leaf explants had been reported to produce callus.…”

Perbanyakan Massa Anggrek Dendrobium Gradita 10 Secara In Vitro Melalui Embriogenesis Somatik

“…Proliferasi kalus dilakukan pada enam komposisi media, yaitu Pro-D1 (ID-1 + 10% air kelapa) ( (Kuo et al 2005). Kalus diproliferasi dalam erlenmeyer 100 ml (Pyrex IWAKI TE-32, Indonesia) dengan kepadatan 1 g kalus/25 ml media.…”