Direct regeneration of protocorm-like bodies (PLBs) from leaf apices of Oncidium flexuosum Sims (Orchidaceae)

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1 DOI /s RESEARCH NOTE Direct regeneration of protocorm-like bodies (PLBs) from leaf apices of Oncidium flexuosum Sims (Orchidaceae) Juliana Lischka Sampaio Mayer Giulio Cesare Stancato Beatriz Appezzato-Da-Glória Received: 24 March 2010 / Accepted: 10 June 2010 Ó Springer Science+Business Media B.V Abstract The present study describes the direct regeneration of protocorm-like bodies (PLBs) in leaf explants of the tropical species Oncidium flexuosum. The explants were inoculated in a solid, modified Murashige and Skoog (MS) medium with different concentrations of the growth regulator thidiazuron (TDZ) and with or without 2,4- dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA), and kept away from light or in a 16-h photoperiod. The presence of auxins, 2,4-D, and NAA inhibited the formation of PLBs. The highest frequency of explants that regenerated PLBs (80%) was obtained when they were maintained in a culture medium containing 1.5 lm TDZ under dark conditions. In the same culture medium but under a 16-h photoperiod, 95% of the leaf explants presented necrosis. Therefore, darkness was crucial for the regeneration of PLBs in O. flexuosum leaf explants, which is in disagreement with the literature. PLBs developed from the division of epidermal and subepidermal cells mainly on the adaxial side of the apex region of the explant. Plants with well-developed leaves and roots grew after the PLBs were transferred to growth regulatorfree medium under a 16-h photoperiod. Keywords Oncidium flexuosum Sims Anatomy Micropropagation Orchid Thidiazuron J. L. S. Mayer B. Appezzato-Da-Glória (&) Biological Sciences Department, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, P.O. Box 09, Piracicaba, São Paulo, Brazil bagloria@esalq.usp.br G. C. Stancato Horticultural Center, Instituto Agronômico, P.O. Box 28, Campinas, São Paulo, Brazil The genus Oncidium Sw. sensu lato is comprised of more than 400 species (Chase et al. 2009), as well as natural and cultivated hybrids, such as ornamental flowers, that possess economical importance as potted plants and cut flowers (Chen and Chang 2000a). O. flexuosum Sims belongs to the sub-family Epidendroideae and occurs naturally in Argentina and Brazil (Pabst and Dungs 1977). There are two known domestic cultivars, one which blossoms during spring (in November and December) and another that starts to bloom during autumn (between April and May) (Mathias 2007). After germination of the orchid seeds, these plants develop into a structure called the protocorm, which has a hypocotyl-like structure of seedlings of different angiosperms (Clements 1995; Cribb 1999). According to Arditti and Ernst (1993), explants such as the tissues or callus of orchids form structures similar to protocorms in vitro; these structures are known as protocorm-like bodies or PLBs. The process of direct or indirect embryogenesis in orchids occurs throughout the formation of PLBs (Arditti and Ernst 1993; Begum et al. 1994; Zhao et al. 2008). For the genus Oncidium, several protocols of micropropagation have been described that involve the formation of embryos or PLBs from young leaves of Oncidium Gower Ramsey (Chen et al. 1999; Chen and Chang 2001, 2002, 2003a, b, 2004; Hong et al. 2008a). Callus proliferation from leaves, stems, and roots, as well as shoot apices of Oncidium Gower Ramsey was reported by Chen and Chang (2000a), Jheng et al. (2006), and Wu et al. (2004). The regeneration of PLBs of Oncidium Sharry Baby OM8 (Li et al. 2005) and Oncidium Sweet Sugar was also reported (Chen and Chang 2000b; Su et al. 2006; Hong et al. 2008a). Such studies have focused only on three hybrids of the genus Oncidium, and the only reports of the regeneration of PLBs in a species were for O. varicosum (Kerbauy 1984, 1993).

2 Due to the demand of O. flexuosum as a cut flower, its breeding has been carried out with the aim of producing cultivars that might blossom between the aforementioned seasons (Mathias 2007). The main goal of the present study was to establish a protocol for micropropagation from leaf explants of O. flexuosum Sims that was aimed at the formation of PLBs to help supply flower market demand as well as breeding programs. According to results previously obtained with Oncidium cultivars, thidiazuron (TDZ) showed the highest ability among cytokinins to promote the formation of PLBs from leaf explants (Chen et al. 1999; Chen and Chang 2001); thus, various concentrations of TDZ have been tested in leaf explants of O. flexuosum. The clonal propagation of orchids by apex leaf cultures is a well-established technique and is less destructive than shoot-tip culture (Churchill et al. 1973; Chen et al. 1999). Artificial pollination was conducted in ten cultivated individuals of O. flexuosum that blossomed during springtime. The voucher of the specimen (150303) was deposited in the UEC Herbarium, Brazil. Mature fruits were kept immersed in 2.5% sodium hypochloride for 20 min and were then rinsed with distilled water. Later, these fruits were sectioned under aseptic conditions in a laminar flow hood and the seeds were directly inoculated in a solid culture medium. The medium used was modified Murashige and Skoog (MS) (1962), with half the concentration of macro and micronutrients and supplementation with 30 g L -1 of sucrose, 5 g L -1 agar (SigmaÒ) and nicotinic acid (0.5 mg L -1 ), pyridoxine HCl (0.5 mg L -1 ), thiamine HCl (0.1 mg L -1 ), glycine (2.0 mg L -1 ), and myo-inositol (100 mg L -1 ), with the ph adjusted to 5.8 using NaOH or HCl 0.1 N before autoclaving. All cultures were maintained in a growth room at 25 ± 2 C under a photoperiod of 16 h and an irradiance of 40 lmol m -2 s -1. White-light lamps were used (PhilipsÒ 32 W/64RS) at a distance of 30 cm from all of the test tubes. Leaf apices (0.5 cm in length) were obtained from 4-month-old plants and used as explants. The explants were inoculated with the adaxial surface in contact with the solid culture medium. The medium used was the same modified MS. Before autoclaving, different concentrations of TDZ, 2,4-dichlorophenoxyacetic acid (2,4-D), and naphthalene acetic acid (NAA) were added to the culture medium. Two light conditions were tested (darkness and 16-h photoperiod). In vitro culture conditions were the same as those described for seed germination. The experimental design was entirely randomized with a factorial scheme (20 combinations of growth regulator and two light conditions) and four replications of five explants for each treatment. Variances among treatments were evaluated according to their homogeneity via Bartlett s test and the treatment means were compared using a Tukey test at a 5% confidence level. The following parameters were evaluated at 90 days after the beginning of the experiment: frequency of explants with regenerating PLBs, mean number of PLBs formed per explant, and mean number of necrotic explants. Explants treated with TDZ that regenerated PLBs were transferred to growth regulator-free medium under a 16-h photoperiod as described above. After 20, 40, and 60 days, the number of PLBs and regenerated plants were counted together because of the difficulty in separating them at this stage of development. For light microscopy analysis, samples of O. flexuosum explants regenerating into PLBs were fixed in Karnovsky (Karnovsky 1965; modified by preparation in phosphate buffer ph 7.2) for 24 h, dehydrated in a graded ethanol series, and embedded in Leica HistoresinÒ (Heraeus Kulzer GmbH and Co. KG, Hanau, Wehrheim, Germany). Serial sections (5-lm thick) were cut on a rotary microtome, stained with toluidine blue O (Sakai 1973), and mounted on Entellan synthetic resin (MerckÒ). Photomicrographs were taken with a LeicaÒ DM LB photomicroscope equipped with a LeicaÒ DC 300F camera (Leica Microsystems Wetzlar GmbH). For scanning electron microscope analyses, samples of O. flexuosum were fixed in Karnovsky (Karnovsky 1965; modified by preparation in phosphate buffer ph 7.2) for 24 h, dehydrated in a graded ethanol series, and critical point-dried with CO 2 (Horridge and Tamm 1969). Samples were attached to aluminum stubs, coated with gold (30 40 nm), and examined under a Carl ZeissÒ LEO VP435 scanning electron microscope at 20 kv. Leaf explants of O. flexuosum showed efficient PLB regeneration but required culture conditions that differed from those described for Oncidium cv. Gower Ramsey and Sweet Sugar (Chen et al. 1999; Su et al. (2006), Vanda (Seeni and Latha 1992), Dendrobium (Chung et al. 2005, 2007), and Phalaenopsis (Kuo et al. 2005). In this study, leaf explants cultivated in a medium free from growth regulators presented a 100% necrosis rate in the presence and absence of light; however, the literature presents contradictory information for the Oncidium Gower Ramsey. According to Chen et al. (1999), this hybrid does not present embryonic explants in a growth regulator-free medium after 30 days of culture. However, Chen and Chang (2003b) showed that the same hybrid presented 40 and 62.5% embryonic explant rates when cultured on growth regulator-free medium for 40 and 60 days, respectively. The presence of TDZ in the culture medium and the absence of light were crucial for the regeneration of PLBs in O. flexuosum (Table 1). TDZ at a concentration of 1.5 lm in the dark condition resulted in an 80% rate of PLB regeneration. This treatment also provided the highest

3 Table 1 Effect of thidiazuron (TDZ), with or without 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA), on the regeneration of protocorm-like bodies (PLBs) from leaf explants of Oncidium flexuosum Sims after 90 days of culture under dark conditions and under a photoperiod of 16 h Concentration (lm) Explants regenerating PLBs (%) Number of PLBs per explant Necrotic explants (%) TDZ 2,4-D NAA Light Dark Light Dark Light Dark Aa 0 Ea 0 Ba 0 Ea 100 Aa 100 Aa Aa 5 Ea 0 Ba 0.25 Ea 100 Aa 75 ABCb Aa 0 Ea 0 Ba 0 Ea 100 Aa 100 Aa Aa 0 Ea 0 Ba 0 Ea 100 Aa 100 Aa Aa 0 Ea 0 Ba 0 Ea 100 Aa 100 Aa Ab 80 Aa 4 Ab 10.8 Aa 90 Aa 15 EFb Ab 10 DEa 0 Ba 1.9 DEa 90 Aa 70 BCb Aa 0 Ea 0 Ba 0 Ea 100 Aa 90 ABa Ab 15 DEa 0 Ba 1.25 Ea 100 Aa 20 EFb Ab 25 CDa 2.3 ABa 2.6 CDEa 80 Aa 10 EFb Ab 70 Aa 0 Bb 7.43 ABa 95 Aa 10 EFb Ab 10 DEa 0 Ba 1.9 DEa 90 Aa 50 CDb Aa 0 Ea 0 Ba 0 Ea 100 Aa 90 ABa Ab 70 Aa 1.8 ABb 5.2 BCDa 90 Aa 10 EFb Ab 35 BCa 0 Bb 3.6 CDEa 95 Aa 10 EFb Ab 50 Ba 0 Bb 3.6 CDEa 90 Aa 10 EFb Ab 15 DEa 0 Ba 1.8 DEa 90 Aa 65 BCb Aa 5 Ea 0 Ba 0.25 Ea 95 Aa 90 ABa Ab 80 Aa 0 Bb 5.9 BCa 90 Aa 5 Fb Ab 40 BCa 1.3 ABab 2.9 CDEa 90 Aa 35 DEb Means followed by the same capital letter within a column and by the same lower-case letter within a row are not significantly different at P B 0.05 mean number of PLBs (10.8) by explant and presented a low frequency of necrotic explants. TDZ combined with NAA also presented an 80% PLB regeneration rate, but with only 5.9 PLBs per explant. On the other hand, the combination of TDZ and 2,4-D under the 16-h photoperiod did not present explants that formed PLBs. In the dark condition, 15% was the maximum frequency of explants with regenerating PLBs. The explants that formed PLBs with TDZ concentrations of 1.5, 4.5, and 13.5 lm and which were maintained under dark conditions were also transferred to a culture medium free from growth regulators and kept under the 16-h photoperiod. Evaluations carried out after 20, 40, and 60 days revealed that some of the PLBs continued to generate secondary PLBs (Fig. 1g, h, arrows), while some PLBs developed into plants (Fig. 1g, h). The explants treated with 1.5 lm TDZ showed an increase in the number of PLBs from 10.8 at the first evaluation of explants kept in the dark to 29.3 (PLBs and regenerated plants) after 60 days in media without growth regulators and under a photoperiod of 16 h (Table 2). The light condition was very important for the efficiency of the protocol in O. flexuosum (Table 1). TDZ at a concentration of 1.5 lm in the presence of light resulted in an explant necrosis rate of 90%. Meanwhile, explants receiving the same treatment but kept in the dark presented a necrosis rate of only 15%. Differing from O. flexuosum, Dendrobium Chiengmai Pink, which presented a higher frequency of embryonic explants and a higher number of embryos with exposure to light (Chung et al. 2007). Recently, Gow et al. (2009) tested the effect of the light regime on the regeneration of Phalaenopsis amabilis (L.) Blume and Phalaenopsis Nebula. Exposure to light delayed the embryogenesis, and the explants tended to become necrotic in both species. Chen and Chang analyzed the embryogenesis in Oncidium (2000b) and Phalaenopsis amabilis (2006), and Chen et al. (1999) described methodology in which explants were maintained under light conditions for 16 h; however, image analysis revealed that newly formed embryos are non-chlorophyllated because they were kept in the dark. Therefore, the dubious description of the culture conditions in the aforementioned studies impedes in vitro protocol follow-through. PLBs originated from epidermal and subepidermal cell divisions (Fig. 1a, b) only at the adaxial face at the explant apex (Fig. 1c e). The same process was observed in

4 Fig. 1 Protocorm-like bodies (PLBs) and plant regeneration from Oncidium flexuosum Sims. a, b Longitudinal sections of the leaf explant. a Cellular divisions of epidermal (ep) and subepidermal layers. b PLBs at the beginning of the development (arrow). c Leaf explant maintained for 90 days in the culture medium under a 16-h photoperiod. d Leaf explant kept under dark conditions after 90 days of culture. e Electron micrograph of PLBs (arrow) after 90 days of culture under dark conditions. f, g, h PLBs (arrows) and plants after 20, 40, and 60 days, respectively, in the culture medium without growth regulator under a 16-h photoperiod; development of the first leaves (l) and roots (r). Bars: (a) = 50 lm; (b) = 100 lm; (c), (d) = 1.25 mm; (e) = 0.5 mm; (f), (g), (h) = 2.5 mm Table 2 Mean number of PLBs and regenerated plants from explants of O. flexuosum Sims after explant transfer to Murashige and Skoog (MS) culture medium that was free of growth regulators and incubation under a 16-h photoperiod TDZ (lm)* 20 days 40 days 60 days a 20.2 a 29.3 a a 15.1 b 25.3 b a 10.9 b 17.6 c * Treatment of origin: TDZ lm kept in the dark. Means followed by the same lower-case letter within a column are not significantly different at P B 0.05 Oncidium Gower Ramsey (Chen and Chang 2001, 2002, 2004) and in Oncidium Sweet Sugar (Su et al. 2006). Churchill et al. (1973) stated that the presence of meristematic tissue at the leaf apex reflects a characteristic that is inherent to the Orchidaceae family and can or cannot be stimulated by the properties of the culture medium. The few explants that regenerated PLBs in the presence of light had a brownish color (Fig. 1c), while explants kept in darkness remained green and produced PLBs without chlorophyll (Fig. 1d). After the transfer of the leaf explants to the culture medium without growth regulator and incubation in the presence of light, the PLBs became green and initiated the development of the first leaves (Fig. 1f, g). During this phase, several PLBs easily disconnected from the explant and remained loose in the culture medium. After 60 days, it was possible to observe the development of plants from the PLBs; these plants presented well-developed leaves and roots (Fig. 1h). The experiment was evaluated at 90 days of culture because, at 60 days, the PLBs appeared as a mass of structures, making them impossible to count, even with a stereomicroscope. However, Chen and Chang (2004) and Jheng et al. (2006), studying Oncidium Gower Ramsey, counted 204 embryos for each leaf explant and more than 1,000 PLBs generated from callus, respectively, after 60 days of culture.

5 In the studied species, the highest frequency of explant regeneration into PLBs was obtained using a low concentration of TDZ (1.5 lm). However, Park et al. (2002) tested different cytokinins in leaf explants of Doritaenopsis, an artificial hybrid between Doritis and Phalaenopsis, and concluded that the best concentration of TDZ for stimulation of the formation of PLBs was 9 lm. Chung et al. (2005) and Kuo et al. (2005) obtained similar results in leaf explants of Dendrobium and Phalaenopsis using and lm TDZ, respectively. The same authors observed that auxins produced an inhibitory effect. Exogenous auxins such as 2,4-D, NAA, indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA) have been reported to have inhibitory effects in explants of Oncidium Gower Ramsey in the genus Oncidium (Chen and Chang 2001). We noticed that 2,4-D alone or in combination with TDZ in the culture medium inhibited the regeneration of PLBs in leaf explants of O. flexuosum. On the other hand, 0.25 lm NAA combined with 13.5 lm TDZ resulted in PLB regeneration in 80% of explants. According to Chen and Chang (2004), the role of endogenous auxins in Oncidium Gower Ramsey has not been completely elucidated to date. These authors obtained the highest frequency of PLB-regenerating leaf explants by using 0.1 and 0.5 lm 2,3,5-triiodobenzoic acid (TIBA), an inhibitor of auxin polar transport. TDZ combined with 2,4-D induces the formation of calli in different genus of Orchidaceae, such as Cymbidium (Chang and Chang 1998), Phalaenopsis (Chen et al. 2000), Paphiopedilum (Lin et al. 2000; Hong et al. 2008b), and Oncidium (Chen and Chang 2000a, b; Jheng et al. 2006). However, the formation of calli in O. flexuosum did not take place for any combination of TDZ with 2,4-D or with NAA. 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