Plbs) from Leaf Apices of Oncidium Flexuosum Sims (Orchidaceae
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Plant Cell Tiss Organ Cult DOI 10.1007/s11240-010-9782-9 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-Glo´ria Received: 24 March 2010 / Accepted: 10 June 2010 Ó Springer Science+Business Media B.V. 2010 Abstract The present study describes the direct regen- The genus Oncidium Sw. sensu lato is comprised of more eration of protocorm-like bodies (PLBs) in leaf explants of than 400 species (Chase et al. 2009), as well as natural and the tropical species Oncidium flexuosum. The explants cultivated hybrids, such as ornamental flowers, that possess were inoculated in a solid, modified Murashige and Skoog economical importance as potted plants and cut flowers (MS) medium with different concentrations of the growth (Chen and Chang 2000a). O. flexuosum Sims belongs to the regulator thidiazuron (TDZ) and with or without 2,4- sub-family Epidendroideae and occurs naturally in dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic Argentina and Brazil (Pabst and Dungs 1977). There are acid (NAA), and kept away from light or in a 16-h pho- two known domestic cultivars, one which blossoms during toperiod. The presence of auxins, 2,4-D, and NAA inhib- spring (in November and December) and another that starts ited the formation of PLBs. The highest frequency of to bloom during autumn (between April and May) (Mathias explants that regenerated PLBs (80%) was obtained when 2007). After germination of the orchid seeds, these plants they were maintained in a culture medium containing develop into a structure called the protocorm, which has a 1.5 lM TDZ under dark conditions. In the same culture hypocotyl-like structure of seedlings of different angio- medium but under a 16-h photoperiod, 95% of the leaf sperms (Clements 1995; Cribb 1999). explants presented necrosis. Therefore, darkness was cru- According to Arditti and Ernst (1993), explants such as cial for the regeneration of PLBs in O. flexuosum leaf the tissues or callus of orchids form structures similar to explants, which is in disagreement with the literature. PLBs protocorms in vitro; these structures are known as proto- developed from the division of epidermal and subepider- corm-like bodies or PLBs. The process of direct or indirect mal cells mainly on the adaxial side of the apex region of embryogenesis in orchids occurs throughout the formation the explant. Plants with well-developed leaves and roots of PLBs (Arditti and Ernst 1993; Begum et al. 1994; Zhao grew after the PLBs were transferred to growth regulator- et al. 2008). free medium under a 16-h photoperiod. For the genus Oncidium, several protocols of micro- propagation have been described that involve the formation Keywords Oncidium flexuosum Sims Á Anatomy Á of embryos or PLBs from young leaves of Oncidium Gower Micropropagation Á Orchid Á Thidiazuron 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), & J. L. S. Mayer Á B. Appezzato-Da-Glo´ria ( ) Jheng et al. (2006), and Wu et al. (2004). The regeneration of Biological Sciences Department, Escola Superior de Agricultura Luiz de Queiroz, Universidade de Sa˜o Paulo, P.O. Box 09, PLBs of Oncidium Sharry Baby ‘OM8’ (Li et al. 2005) and 13418-900 Piracicaba, Sa˜o Paulo, Brazil Oncidium Sweet Sugar was also reported (Chen and Chang e-mail: [email protected] 2000b; Su et al. 2006; Hong et al. 2008a). Such studies have focused only on three hybrids of the genus Oncidium, and G. C. Stancato Horticultural Center, Instituto Agronoˆmico, P.O. Box 28, the only reports of the regeneration of PLBs in a species 13012-970 Campinas, Sa˜o Paulo, Brazil were for O. varicosum (Kerbauy 1984, 1993). 123 Plant Cell Tiss Organ Cult Due to the demand of O. flexuosum as a cut flower, its Bartlett’s test and the treatment means were compared breeding has been carried out with the aim of producing using a Tukey test at a 5% confidence level. cultivars that might blossom between the aforementioned The following parameters were evaluated at 90 days seasons (Mathias 2007). after the beginning of the experiment: frequency of The main goal of the present study was to establish a explants with regenerating PLBs, mean number of PLBs protocol for micropropagation from leaf explants of formed per explant, and mean number of necrotic explants. O. flexuosum Sims that was aimed at the formation of PLBs Explants treated with TDZ that regenerated PLBs were to help supply flower market demand as well as breeding transferred to growth regulator-free medium under a 16-h programs. photoperiod as described above. After 20, 40, and 60 days, According to results previously obtained with Oncidium the number of PLBs and regenerated plants were counted cultivars, thidiazuron (TDZ) showed the highest ability together because of the difficulty in separating them at this among cytokinins to promote the formation of PLBs from stage of development. leaf explants (Chen et al. 1999; Chen and Chang 2001); For light microscopy analysis, samples of O. flexuosum thus, various concentrations of TDZ have been tested in explants regenerating into PLBs were fixed in Karnovsky leaf explants of O. flexuosum. The clonal propagation (Karnovsky 1965; modified by preparation in phosphate of orchids by apex leaf cultures is a well-established buffer pH 7.2) for 24 h, dehydrated in a graded ethanol technique and is less destructive than shoot-tip culture series, and embedded in Leica HistoresinÒ (Heraeus Kul- (Churchill et al. 1973; Chen et al. 1999). zer GmbH and Co. KG, Hanau, Wehrheim, Germany). Artificial pollination was conducted in ten cultivated Serial sections (5-lm thick) were cut on a rotary micro- individuals of O. flexuosum that blossomed during spring- tome, stained with toluidine blue O (Sakai 1973), and time. The voucher of the specimen (150303) was deposited mounted on Entellan synthetic resin (MerckÒ). Photomi- in the UEC Herbarium, Brazil. Mature fruits were kept crographs were taken with a LeicaÒ DM LB photomicro- immersed in 2.5% sodium hypochloride for 20 min and scope equipped with a LeicaÒ DC 300F camera (Leica were then rinsed with distilled water. Later, these fruits Microsystems Wetzlar GmbH). were sectioned under aseptic conditions in a laminar flow For scanning electron microscope analyses, samples of hood and the seeds were directly inoculated in a solid O. flexuosum were fixed in Karnovsky (Karnovsky 1965; culture medium. The medium used was modified Murash- modified by preparation in phosphate buffer pH 7.2) for ige and Skoog (MS) (1962), with half the concentration of 24 h, dehydrated in a graded ethanol series, and critical macro and micronutrients and supplementation with 30 g point-dried with CO2 (Horridge and Tamm 1969). Samples L-1 of sucrose, 5 g L-1 agar (SigmaÒ) and nicotinic acid were attached to aluminum stubs, coated with gold (30– (0.5 mg L-1), pyridoxine HCl (0.5 mg L-1), thiamine HCl 40 nm), and examined under a Carl ZeissÒ LEO VP435 (0.1 mg L-1), glycine (2.0 mg L-1), and myo-inositol scanning electron microscope at 20 kV. (100 mg L-1), with the pH adjusted to 5.8 using NaOH or Leaf explants of O. flexuosum showed efficient PLB HCl 0.1 N before autoclaving. All cultures were main- regeneration but required culture conditions that differed tained in a growth room at 25 ± 2°C under a photoperiod from those described for Oncidium cv. Gower Ramsey and of 16 h and an irradiance of 40 lmol m-2s-1. White-light Sweet Sugar (Chen et al. 1999; Su et al. (2006), Vanda lamps were used (PhilipsÒ 32 W/64RS) at a distance of (Seeni and Latha 1992), Dendrobium (Chung et al. 2005, 30 cm from all of the test tubes. Leaf apices (0.5 cm in 2007), and Phalaenopsis (Kuo et al. 2005). length) were obtained from 4-month-old plants and used as In this study, leaf explants cultivated in a medium free explants. from growth regulators presented a 100% necrosis rate in the The explants were inoculated with the adaxial surface in presence and absence of light; however, the literature pre- contact with the solid culture medium. The medium used sents contradictory information for the Oncidium Gower was the same modified MS. Before autoclaving, different Ramsey. According to Chen et al. (1999), this hybrid does concentrations of TDZ, 2,4-dichlorophenoxyacetic acid not present embryonic explants in a growth regulator-free (2,4-D), and naphthalene acetic acid (NAA) were added to medium after 30 days of culture. However, Chen and Chang the culture medium. Two light conditions were tested (2003b) showed that the same hybrid presented 40 and (darkness and 16-h photoperiod). In vitro culture conditions 62.5% embryonic explant rates when cultured on growth were the same as those described for seed germination. regulator-free medium for 40 and 60 days, respectively. The experimental design was entirely randomized with a The presence of TDZ in the culture medium and the 20 9 2 factorial scheme (20 combinations of growth reg- absence of light were crucial for the regeneration of PLBs ulator and two light conditions) and four replications of in O. flexuosum (Table 1). TDZ at a concentration of five explants for each treatment. Variances among treat- 1.5 lM in the dark condition resulted in an 80% rate of ments were evaluated according to their homogeneity via PLB regeneration.