Plant regeneration via callus culture and subsequent in vitro flowering of Dendrobium huoshanense

Acta Physiol Plant (2014) 36:2619 2625 DOI 10.1007/s11738-014-1632-7 ORIGINAL PAPER Plant regeneration via callus culture and subsequent in vitro flowering of Dendrobium huoshanense Po-Lun Lee Jen-Tsung Chen Received: 26 December 2013 / Revised: 17 June 2014 / Accepted: 2 July 2014 / Published online: 22 July 2014 Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2014 Abstract A protocol for regenerating and subsequent in vitro flowering of an economical important and endangered medicinal orchid, Dendrobium huoshanense, was established mainly via indirect protocorm-like body (PLB) formation. A four-step method was developed to induce successful plant regeneration on 1/2 MS medium supplemented with suitable plant growth regulators (PGRs). Step 1 (callus induction): the root tip explants (1 cm long) were cultured at 1 mg l -1 2,4-D? 1mgl -1 TDZ for 3 months. Step 2 (callus proliferation): the calli were subcultured with a 1-month interval at 1 mg l -1 2,4-D? 1mgl -1 TDZ. Step 3 (PLB induction): the calli were cultured at 2 mg l -1 NAA? 1mgl -1 BA for 2 months. Step 4 (plantlet conversion): the 2-month-old PLBs were cultured at 0.1 mg l -1 IBA for 4 months. It took at least 6 months to produce well-rooted regenerated plantlets with an average of 3.2 roots and 3.6 leaves from the initial callus. The 6-month-old rooted plantlets were transferred onto PGRfree 1/2 MS medium for 6 months, and then potted with Sphagnum moss for acclimatization. After 2 month of culture, the survival rate was 100 %. The in vitro flowers were obtained on the 8-month-old plantlets at 1 mg l -1 IBA, 5 mg l -1 IBA and 0.1 mg l -1 NAA, but the flowers showed a lack of the gynandrium. The abnormity was overcome by the aid of 5 mg l -1 TDZ, and subsequently, the capsules formed without artificial pollination. This protocol provides the basis for further investigation on cell Communicated by B. Borkowska. P.-L. Lee J.-T. Chen (&) Department of Life Sciences, National University of Kaohsiung, Kaohsiung 811, Taiwan, Republic of China e-mail: jentsung@nuk.edu.tw suspension, micropropagation, in vitro flowering and breeding programs in Dendrobium huoshanense. Keywords Callus culture Cytokinin Orchid Protocorm-like body Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid 2iP N 6-2-Isopentenyl adenine BA N 6 -Benzyladenine IBA Indole-3-butyric acid Kinetin N 6 -Furfuryladenine MS Murashige and skoog (1962) medium NAA 1 Naphthaleneacetic acid PGR Plant growth regulator PLB Protocorm-like body TDZ 1-Phenyl-3-(1,2,3-thiadiazol-5-yl)-urea, thidiazuron Introduction Dendrobium huoshanense (Orchidaceae), commonly known as thousand gold piece herb or soft gold, is very important to traditional Chinese medicine (Tang and Cheng 1984; Bao et al. 2001). The dried stem is the primary part of D. huoshanense and it contains medicinal constituents, including flavonoids, polysaccharides, and alkaloids (Tang and Cheng 1984; Bao et al. 2001; Chang et al. 2010). It had been documented in Shennong s Herbal as a high-grade drug, and was used for thousands of years in Chinese medicine for curing fever, throat inflammation, adjusting eye and body function, easing the internal heat of the body and prolonging life (Tang and Cheng 1984; Bao et al. 2001; Chang et al. 2010). Due to the high economic value

2620 Acta Physiol Plant (2014) 36:2619 2625 of stems, wild plants of D. huoshanense have been harvested excessively. Unfortunately, long-term harvesting caused the acute habitat destruction and this species is endangered which subjected to Annex II of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora). In vitro culture provides an alternative way for recovery of this endangered species and it could also provide sources of the herbal drug (Luo et al. 2003, 2009). It also served as a platform for inducing suspension cell culture, somaclonal variation, and obtaining transgenic plants, etc., to aid plant breeding programs (Pedroso and Pais 1995). However, there were limited successes on tissue culture protocols of D. huoshanense, and the previous protocols mainly developed for asymbiotic germination from immature or mature seeds or inducing shoot elongation from stem nodes or protocormlike bodies (PLBs, structures raised from in vitro explants and resembled to seed-derived protocorms) (Luo et al. 2003, 2009; Wang et al. 2006; Jiang et al. 2006; Jin et al. 2008). To our best knowledge, this study is the first to establish a complete protocol for inducing callus formation, PLB formation, plantlet conversion, and in vitro flowering in this economic important and endangered medicinal plant. Materials and methods Culture medium The basal medium (BM) used in all the experiments consisted of 1/2 strength of MS (Murashige and Skoog 1962 medium), 20 g l -1 sucrose, 1 g l -1 peptone, 4 g l -1 Gelrite and supplemented with various PGRs (as the experimental design). The ph of media was adjusted to 5.2 with 1 M KOH or HCl prior to autoclaving for 20 min at 121 C. Plant materials The seed-derived protocorms of D. huoshanense C. Z. Tang and S. J. Cheng were purchased from a local orchid farm, Nantou, Taiwan. These protocorms were cultured on hormone-free BM in glass bottles (600 ml). All the cultures were incubated in a growth chamber under a 16/8-h (light/dark) photoperiod at irradiance of 42 55 lmol m -2 s -1 (daylight fluorescent tubes FL- 20BR/18, 18 W, China Electric Co., Taipei, Taiwan) and temperature of 25 ± 2 C. The subculture period was 6 weeks. Protocorm-derived plants (about 2 cm height) with several leaves and roots were used as donor plants in this present work. Callus induction Root tip segments (1 cm long), leaf tip segments (1 cm long), and stem nodal segments (each with 1 node) taken from the donor plants were used to induce callus formation on BM supplemented with combinations of 2,4-D (0, 1, 2, 5 and 10 mg l -1 ) and TDZ (0, 0.1, 0.5 and 1 mg l -1 ). All the explants were placed on the culture media and incubated in 18 9 150 mm 2 test tubes in darkness for 12 weeks with a 4-week interval subculture. Six replicates (tubes) were provided for each treatment. The percentages of explants producing callus were counted under a stereomicroscope (SZH, Olympus, Tokyo, Japan) and were recorded after 12 weeks of culture. The callus lines were referred as XD y T z (X = R means root-derived, L means leaf-derived, S means stem-derived, D y means 2,4-D at y mg l -1,T z means TDZ at z mg l -1 ). Callus proliferation and PLB induction A callus line RD 1 T 1, derived from root tip explants on BM? 1mgl -1 2,4-D? 1mgl -1 TDZ, proliferated more and stayed viable on the same medium in darkness. Callus masses (each was 0.1 g in fresh weight) taken from the 1-year-long proliferated (with a 1-month interval of subculture) callus were used as explants to induce PLB formation on BM supplemented with combinations of NAA (0, 0.5, and 2 mg l -1 ) plus TDZ (0, 0.3, 1, and 3mgl -1 ), or NAA (0, 0.5, and 2 mg l -1 ) plus BA (0, 0.3, 1, and 3 mg l -1 ). All the calli were placed on the culture media and incubated in 18 9 150 mm 2 test tubes in darkness for 2 months with a 1-month-interval subculture. Five replicates (tubes) each contained one callus mass were provided for each treatment. The percentages of explants producing PLBs were counted under a stereomicroscope and were recorded after 2 months of cultivation. Root induction and in vitro flowering from PLBs Two-month-old callus-derived PLBs, induced at 2 mg l -1 NAA? 1mgl -1 BA, could be easily detached from the parent callus masses. These PLBs were transferred onto BM supplemented with IBA (0, 0.1, 1, and 5 mg l -1 )or NAA (0, 0.1, 1, and 5 mg l -1 ) to induce root formation and subsequent in vitro flowering. All the PLBs were placed on the culture media and incubated in 18 9 150 mm 2 test tubes under a light condition with 16/8-h (light/dark) photoperiod, irradiance of 42 55 lmol m -2 s -1 and temperature of 25 ± 2 C. The subculture period is 1 month. Six replicates (tubes) each contained one PLB were provided for each treatment. Number of roots per explant and the response of in vitro flowering were recorded after 4 and 6 months of culture, respectively.

Acta Physiol Plant (2014) 36:2619 2625 2621 Effects of cytokinins on in vitro flowering Two-month-old PLBs (4 5 mm in diameter) were used to test the effects of cytokinins on in vitro flower formation on 0.5 mg l -1 NAA-containing BM supplemented with 0, 0.1, and 5 mg l -1 2iP, BA, kinetin, or TDZ. All the cultures were kept in a light condition as mentioned above and the subculture period is 4 weeks. Six replicates (tubes) each contained one PLB were provided for each treatment. Number of in vitro flowers per plantlet was recorded after 6 months of culture. Plantlet development and acclimatization Six-month-old viable plantlets induced on BM? 0.1 mg l -1 NAA were transferred onto PGR-free BM in 600 ml flasks with a 1-month-interval subculture period for 6 months. The light, photoperiod, and temperature regimes were the same as above. Subsequently, these plantlets were transferred onto three-inch plastic pots with Sphagnum moss for acclimatization. Data analysis The data on all the parameters were subjected to one-way analysis of variance (ANOVA). The data expressed as percentages were transformed using arcsine prior to ANOVA and then converted back to the original scale (Compton 1994). All treatment means were compared by following Duncan s multiple range test. Significant differences between means were presented at the level of P B 0.05. Results Callus induction and proliferation When the explants were cultured on media for 4 weeks in darkness, pale whitish tissue swelling was found on the root tip explants at 1 mg l -1 2,4-D, 1 mg l -1 2,4- D? 0.5 mg l -1 TDZ and 1 mg l -1 2,4-D? 1mgl -1 TDZ, and the stem nodal explants at 1 mg l -1 2,4- D? 0.1 mg l -1 TDZ and 2 mg l -1 2,4-D? 0.1 mg l -1 TDZ (data not shown). After another 2 months of cultivation, the swelled tissues developed into yellowish callus Table 1 Effects of 2,4-D and TDZ on percentages of callus formation from root tip explants of Dendrobium huoshanense 2,4-D TDZ Explant type Leaf segment Root tip segment Stem node 0 0 0 0 b 0 a 1 0.1 0 0 b 17 a 1 0.5 0 17 ab 0 a 1 1 0 33 a 0 a Fig. 1 Callus formation and plant regeneration of Dendrobium huoshanense. a Yellowish callus formed from the tip of a root explant on 1/2MS? 1mgl -1 2,4- D? 1mgl -1 TDZ after 3 months of culture in darkness (bar 2.5 mm). b Callus proliferated more when transferred on the same medium for 1 year in darkness (bar 5 mm). c PLBs formed when the root tip-derived callus transferred onto 1/2MS? 2mgl -1 NAA? 1mgl -1 BA for 2 months in a light condition with 16/8-h (light/dark) photoperiod (bar 8 mm). d Plantlets established when the 2-month-old PLBs transferred onto PGR-free 1/2MS for 4 months of culture in a light condition with 16/8-h (light/ dark) photoperiod (bar 1.2 cm)

2622 Acta Physiol Plant (2014) 36:2619 2625 from the root tip explants at 1 mg l -1 2,4-D? 0.5 mg l -1 TDZ (callus line RD1T0.5) and 1 mg l -1 2,4- D? 1mgl -1 TDZ (callus line RD1T1), and the stem nodal explants at 1 mg l -1 2,4-D? 0.1 mg l -1 TDZ (callus line SD1T0.1) (Table 1). In contrast, swelled tissues failed to form callus from the root tip explants at 1 mg l -1 2,4-D, and the stem nodal explants at 2 mg l -1 2,4- D? 0.1 mg l -1 TDZ. In these treatments, the swelled tissues of the root tip and the stem nodal explants turned brown and eventually necrosis. The leaf explants had no response on callus induction in all the treatments (Table 1), and these explants became necrosis after 3 months of culture. The highest percentage of explants forming callus was resulted in the root tip explants at 1 mg l -1 2,4- D? 1mgl -1 TDZ (Table 1). All the calli formed were originated from the tip region of the responding root explants (Fig. 1a), and it may be due to heterogeneity of the explants. After 1 year of culture, only callus line RD1T1 proliferated more and maintained the viability (Fig. 1b). In contrast, RD1T0.5 and SD1T0.1 failed to proliferate and became necrosis eventually (data not shown). Induction of PLB formation Five treatments, including 3 mg l -1 TDZ, 0.5 mg l -1 NAA, 0.5 mg l -1 NAA? 0.3 mg l -1 TDZ, 0.3 mg l -1 BA, and 0.5 mg l -1 NAA? 1mgl -1 BA, induced PLBs from the callus masses (Table 2). The callus mass turned greenish in color after several days of culture, then became more compact, and subsequent formed PLBs after 2 months of culture. The most viable PLBs were obtained at 2 mg l -1 NAA? 1mgl -1 BA, and these PLBs grew well and developed the sheath leaves more quickly than others (Fig. 1c). After 2 months of culture, average 4 PLBs could be obtained from 0.1 g of callus mass in this treatment (data not shown). PLB development and in vitro flowering It was found that the callus-derived PLBs had an intrinsic difficulty to form roots (Fig. 1d) and an average of 0.8 roots per plantlet was obtained on PGR-free BM after 4 months of culture (Table 3). In the presence of 1 mg l -1 IBA, 5 mg l -1 IBA, and 0.1 mg l -1 NAA, a significantly increase of root formation were obtained (Table 3). After 4 months of culture, each plantlet formed an average of 3.6 4.0 roots (Table 3). After another 2 months of culture, the plantlets produced viable flowers at 1 mg l -1 IBA, 5mgl -1 IBA, and 0.1 mg l -1 NAA (Table 3; Fig. 2a, b). Although the plantlets occasionally produced flower buds at PGR-free BM, but all were subsequent aborted and did not open eventually (Fig. 2c). It was suggested that without Table 2 Effects of NAA, TDZ, and BA on percentages of PLB formation from root-derived callus of Dendrobium huoshanense PGRs NAA TDZ BA 0 0 0 b 0 0.3 20 ab 0 1 40 ab 0 3 0 b 0.5 0 0 b 0.5 0.3 0 b 0.5 1 40 ab 0.5 3 20 ab 2 0 80 a 2 0.3 60 ab 2 1 40 ab 2 3 40 ab 0 0.3 0 b 0 1 20 ab 0 3 20 ab 0.5 0.3 60 ab 0.5 1 0 b 0.5 3 20 ab 2 0.3 40 ab 2 1 80 a 2 3 60 ab % of explants forming PLBs Table 3 Effects of IBA and NAA on root formation and in vitro flowering from plantlets of Dendrobium huoshanense PGRs IBA NAA No. of roots/ plantlet 0 0 0.8 bc - 0.1 0 3.2 ab - 1 0 3.6 a? 5 0 3.6 a? 0 0.1 4.0 a? 0 1 2.2 abc - 0 5 0 c - Response of in vitro flowering? plantlets with viable in vitro flowers, - plantlets with no viable in vitro flowers well root formation at PGR-free BM, the plantlets had not enough of nutrients to support the entire development process of flowers. The in vitro flowers opened well on the

Acta Physiol Plant (2014) 36:2619 2625 2623 Fig. 2 In vitro flowering of Dendrobium huoshanense. a A flower bud (arrow) formed from a 6-month-old plantlet on 1/2MS? 0.1 mg l -1 NAA (bar 2.5 mm). b A flower bud developed well on 1/2MS? 0.1 mg l -1 NAA (bar 2.5 mm). c Without root formation, the flower bud aborted (arrow) on PGR-free 1/2MS (bar 1 mm). d An abnormal flower without the gynandrium formed from a 8-month-old plantlet on 1/2MS? 0.1 mg l -1 NAA (bar 4 mm). e An well-developed flower with the gynandrium (arrow) formed from a 8-month-old plantlet on 1/2MS? 0.5 mg l -1 NAA? 5 TDZ mg l -1 (bar 5 mm). f A capsule (arrow) formed from a 10-month-old plantlet on 1/2MS? 0.5 mg l -1 NAA? 5 TDZ mg l -1 (bar 5 mm) 8-month-old plantlets at 1 mg l -1 IBA, 5 mg l -1 IBA, and 0.1 mg l -1 NAA, but these flowers showed an abnormity that lack of the gynandrium (Fig. 2d). The longest life span of these in vitro flowers was about 2 weeks at 0.1 mg l -1 NAA, and they were finally necrosis. Effects of cytokinins on in vitro flowering Two-month-old callus-derived embryos, induced at 2mgl -1 NAA? 1mgl -1 BA, were transferred onto 0.5 mg l -1 NAA-containing BM supplemented with various concentrations of cytokinins to enhance in vitro flowering (Table 4). The results showed that 5 mg l -1 TDZ significantly enhanced the number of in vitro flowers per plantlet when compared with the control treatment (Table 4). These in vitro flowers showed normal morphology with a well-developed gynandrium (Fig. 2e), and subsequent formed a capsule without the artificial pollination (Fig. 2f). When compared with the in vitro flowers induced at 1 mg l -1 IBA, 5 mg l -1 IBA, and 0.1 mg l -1 NAA, it indicated that a high dose of TDZ at 5 mg l -1 may play a crucial role in the formation of the gynandrium. Plantlet conversion, growth, and acclimatization Two-month-old callus-derived PLBs, induced at 2 mg l -1 NAA? 1mgl -1 BA, were transferred onto BM supplemented with 0.1 mg l -1 IBA for inducing better root

2624 Acta Physiol Plant (2014) 36:2619 2625 formation without in vitro flowering. After 4 months of cultivation, plantlets grew well and produced an average of 3.2 roots and 3.6 leaves. The rooted plantlets grew well on PGR-free BM (Fig. 3a). After 2 months of acclimatization, the pot plants showed normal growth and morphology with a 100 % of survival rate (Fig. 3b). Discussion Roy et al. (2007) reported that both of callus induction and direct PLB formation from the cut surface of shoot tip explants of Dendrobium chrysotoxum was achieved using TDZ at 1 16 lm (about 0.22 3.52 mg l -1 ). However, in this study, single use of TDZ at 0.1 1 mg l -1 did not induce callus formation or direct PLB formation from root, leaf, and stem nodal explants. It needs to be combined with 2,4-D at 1 mg l -1 for inducing callus formation (Table 1). It may reflect that the morphogenetic flexibility of shoot tip explants was higher than the fully developed vegetative organs. Table 4 Effects of cytokinins on numbers of in vitro flowers from plantlets of Dendrobium huoshanense Cytokinins No. of in vitro flowers/plantlet PGR-free 0 0 b 2iP 0.1 0.2 ab 5 0.2 ab BA 0.1 0 b 5 0.7 ab Kinetin 0.1 0.2 ab 5 0.2 ab TDZ 0.1 0.2 ab 5 1.17 a The leaf explants were successful in inducing direct or indirect somatic embryogenesis in several orchids, including Oncidium, Phalaenopsis, and Dendrobium (Chen et al. 1999; Chung et al. 2005; Kuo et al. 2005; Chen and Chang 2006). Chung et al. (2007) used TDZ to induce direct somatic embryogenesis from the leaf explants of Dendrobium cv. Chiengmai Pink, and the best treatment was at 1 mg l -1. However, it was found that the young leaf explants of D. huoshanense had no morphogenetic response to all the concentrations of TDZ (0.1, 0.5, and 1mgl -1 ). It reflected the low morphogenetic flexibility of the leaf explants of D. huoshanense when compared with the other orchid species. Roy et al. (2007) used 0.22 3.52 mg l -1 TDZ to induce callus that exhibited complete hormone autonomy for proliferation and a spontaneously differentiation of PLBs, and it was a one-step method to accomplish dedifferentiation, proliferation, and redifferentiation. It indicated that the modification of tissue culture protocol was necessary for a better result of in vitro propagating in different orchids. In Dendrobium orchids, in vitro flowering was intensively induced using in vitro seedlings or in vitro regenerated plantlets (Chia and He 1999; Alex et al. 2008; Hee et al. 2007; Sim et al. 2008; Tee et al. 2008; Wang et al. 2009), Sim et al. (2007) induced in vitro seedpod (=capsule) formation via artificial pollination using pollens of in vivo plants and the ovaries of in vitro plants. Some of the seeds had the ability to germinate, but the seed density in seedpod was low (Sim et al. 2007). In this study, the capsules developed spontaneously without artificial pollination on the plantlets at 5 mg l -1 TDZ, and it may due to swollen of the ovaries or even parthenogenesis. Luo et al. (2009) established a two-step method to obtained rooted plantlets from PLBs. Firstly, they induced PLBs to shoot at 20 lm (about 4.3 mg l -1 ) kinetin and 10 g l -1 maltose. Secondary, the shoots were rooted on PGR-free MS medium supplemented with 8 g l -1 banana Fig. 3 Plantlet growth and acclimatization of Dendrobium huoshanense. a One-year-old plantlets were ready for transplanting into pots (bar 2.5 cm). b Regenerated plants grew well after 2 months of culture on a plastic pot with Sphagnum moss (bar 2.5 cm)

Acta Physiol Plant (2014) 36:2619 2625 2625 paste. In this study, the PLBs were converted into wellrooted plantlets in a more efficient way using a one-step method on BM supplemented with 0.1 mg l -1 IBA. Conclusion This study is the first to establish a reliable protocol for regenerating D. huoshanense via indirect PLB formation. It took about 9 months to obtained morphological normal regenerated plantlets from root tip explants via an in vitro morphogenetic pathway that embryogenic callus developed initially (3 months), followed by formation of PLBs (2 months), and then converted into rooted plantlets (4 months). This protocol provided the basis for further investigation on cell suspension, micropropagation, in vitro flowering, or breeding programs in D. huoshanense. Author contribution J. T. Chen conceived the idea and designed the experiments of this work. P. L. Lee and J. T. Chen both conducted the research and J. T. Chen wrote the manuscript. Acknowledgments The authors are very grateful for the financial support from National University of Kaohsiung and Ministry of Science and Technology of ROC. References Alex S, Rajmohan K, John MSG, Soni KB (2008) In vitro flowering of orchids: a tool for early testing of novel varieties. Curr Biot 2:104 109 Bao XS, Shun QS, Chen LZ (2001) The medicinal plants of Dendrobium (Shi-Hu) in China. Fudan University Press, Shanghai, A Coloured Atlas Chang CC, Ku AF, Tseng YY, Yang WB, Fang JM, Wong CH (2010) 6,8-Di-C-glycosyl flavonoids from Dendrobium huoshanense. J Nat Prod 73:229 232 Chen JT, Chang WC (2006) Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis. Biol Plant 50:169 173 Chen JT, Chang C, Chang WC (1999) Direct somatic embryogenesis on leaf explants of Oncidium Gower Ramsey and subsequent plant regeneration. Plant Cell Rep 19:143 149 Chia TF, He J (1999) Review: in vitro flowering of orchids. Lindlenyana 14:60 76 Chung HH, Chen JT, Chang WC (2005) Cytokinins induce direct somatic embryogenesis of Dendrobium Chiengmai Pink and subsequent plant regeneration. In Vitro Plant 41:765 769 Chung HH, Chen JT, Chang WC (2007) Plant regeneration through direct somatic embryogenesis from leaf explants of Dendrobium. Biol Plant 51:346 350 Compton ME (1994) Statistical methods suitable for the analysis of plant tissue culture data. Plant Cell Tiss Org Cult 37:217 242 Duncan DB (1955) Multiple range and multiple F test. Biometrics 11:1 42 Hee KH, Loh CS, Yeoh HH (2007) Early in vitro flowering and seed production in culture in Dendrobium chao praya smile (Orchidaceae). Plant Cell Rep 26:2055 2062 Jiang ST, Wei MM, Luo JP (2006) Effect of phosphate on growth and polysaccharide production by suspension cultures of protocormlike bodies of Dendrobium huoshanense. Chin J Biotech 22:613 618 Jin Q, Ma SJ, Hong SL, Cai YP, Lin Y (2008) Induction of protocorm-like bodies from Dendrobium huoshanense and effects of different culture methods on protocorm multiplication. J Anhui Agric Univ 35:258 261 Kuo HL, Chen JT, Chang WC (2005) Efficient plant regeneration through direct somatic embryogenesis from leaf explants of Phalaenopsis Little Steve. In Vitro Plant 41:453 456 Luo JP, Zha XQ, Jiang ST (2003) Suspension culture of protocormlike bodies from the endangered medicinal plant Dendrobium huoshanenese. China J Chin Mater Med 28:611 614 Luo JP, Wawrosch C, Kopp B (2009) Enhanced micropropagation of Dendrobium huoshanense C. Z. Tang and S. J. Cheng through protocom-like bodies: the effects of cytokinins, carbohydrate source and cold pretreatment. Sci Hortic :258 262 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:495 497 Pedroso MC, Pais MS (1995) Factors controlling somatic embryogenesis. Plant Cell Tiss Org Cult 43:147 154 Roy J, Naha S, Majumdar M, Banerjee N (2007) Direct and callusmediated protocorm-like body induction from shoot-tips of Dendrobium chrysotoxum Lindl (Orchidaceae). Plant Cell Tiss Org Cult 90:31 39 Sim GE, Lon CS, Goh CJ (2007) High frequency early in vitro flowering of Dendrobium Madame Thong-In (Orchidaceae). Plant Cell Rep 26:383 393 Sim GE, Goh CJ, Lon CS (2008) Induction of in vitro flowering in Dendrobium Madame Thong-In (Orchidaceae) seedlings is associated with increase in endogenous ip and ipa levels. Plant Cell Rep 27:1281 1289 Tang ZZ, Cheng SJ (1984) A study on the raw plants for Chinese traditional medicinal Huoshan Shi-hu. Bull Bot Res 4:141 146 Tee SC, Maziah M, Tan CS (2008) Induction of in vitro flowering in the orchid Dendrobium Sonia 17. Biol Plant 52:723 726 Wang Y, Luo JP, Zha XQ (2006) Protocorm-like body formation and plant regeneration of Dendrobium huoshanese, an endangered medicinal plant. Acta Hortic 725:379 384 Wang ZH, Wang L, Ye QS (2009) High frequency early flowering from in vitro seedlings of Dendrobium nobile. Sci Hortic 112:328 331