An efficient direct induction of protocorm-like bodies from leaf subepidermal cells of Doritaenopsis hybrid using thin-section culture

Abstract

High-frequency protocorm-like body (PLB) formation directly from thin leaf sections of Doritaenopsis hybrid was achieved in order to develop a mass-scale propagation system. Concentrated efforts were made to study the effects of different cytokinins on in vitro PLB induction from thin leaf sections. Among the cytokinins tested, thidiazuron (TDZ) was found to be a more effective inducer of PLBs than benzyladenine and zeatin. A modified Murashige and Skoog medium supplemented with 9.0 µM TDZ was found to be the optimum concentration for PLB development from thin leaf sections of Doritaenopsis hybrid. Of the two different explant types used in the present experiment, the highest percentage of PLB formation (72.3%) and highest number of PLBs (18) per explant were observed on thin leaf sections (1 mm thick), while only 20% (4.3 per explant) of comparatively large leaf segments (5 mm thick) were able to produce PLBs under the same culture conditions. Light microscopy observations indicated that the initial cell divisions for PLB formation occurred on the region near the cut surface and that an intact epidermal layer appeared to play an important role in PLB formation. Proembryo initiation occurred from several cells just beneath the intact epidermal cell, and globular PLBs were clearly visible after 3 weeks of culture and subsequently developed into mature PLBs.

Efficient adventitious shoot regeneration from shoot tip culture of Vanda coerulea, a Thai orchid

“…TDZ concentration higher than 0.1 mg/l reduced shoot formation, as has been reported for Doritaenopsis hybrid 24 and Cymbidium sinense 26 . Comparing the effects of BA and TDZ at different levels on shoot formation of V. coerulea, it was found that TDZ can be used at a much lower concentration than BA, in agreement with a previous study 25 . In this study, not only shoots could be promoted from shoot tips, but also roots were formed simultaneously from shoots in all treatments.…”

“…TDZ was efficient to induce direct somatic embryogenesis from leaf explants but it was ineffective to induce somatic embryogenesis from root of Oncidium 21,22 . TDZ alone or in combination with auxins has been used to induce embryogenesis in Cymbidium ensifolium 23 , Oncidium 22 , protocorm-like bodies in Doritaenopsis 24 and Doritaenopsis hybrid 25 . The results suggested that 0.1 mg/l TDZ induced high shoot formation from shoot tips of V. coerulea.…”

Phalaenopsis LEAFY COTYLEDON1-Induced Somatic Embryonic Structures Are Morphologically Distinct From Protocorm-Like Bodies

“…For years, much effort has been devoted to develop in vitro protocols to induce PLB and somatic embryo development either directly or indirectly ( via the callus tissue) from explants to improve micropropagation in Phalaenopsis orchids (Tokuhara and Mii, 2001; Tokuhara and Mii, 2003; Kuo et al, 2005; Chen and Chang, 2006; Gow et al, 2009; Gow et al, 2010; Pramanik et al, 2016). PLBs are often induced from somatic tissues such as protocorms, floral stalk internodes, leaves, and root tips (Chen et al, 2002; Park et al, 2002; Chen and Chang, 2004; Chen and Chang, 2006; Teixeira da Silva et al, 2006; Zhao et al, 2008; Guo et al, 2010; Paek et al, 2011). Because embryogenesis produces tubular embryos, which, upon germination, develop into protocorms, and PLBs resemble protocorms morphologically, initiation, and development of PLBs is often regarded as somatic embryogenesis (Jones and Tisserat, 1990; Begum et al, 1994; Ishii et al, 1998).…”

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).…”