Micropropagation of Dendrobium

Micropropagation or large-scale clonal propagation (i.e., excluding seeds) is the most important (practical and economical) application of plant biotechnology, although care must be taken, for clonal propagation studies, to avoid somaclonal variation (Bairu et al. 2011). Since variation can take place when Dendrobium is propagated by keikis, i.e., root-bearing adventitious growths, in vitro propagation offers the ability to ensure clonal stability (Li et al. 2013a). The induction of totipotent callus, somatic embryogenesis from callus, direct somatic embryogenesis (syn. protocorm-like body, PLB; Fig. 1a) induction (Teixeira da Silva and Tanaka, 2006), direct induction of axillary shoots (Pyati et al. 2002), and direct regeneration through shoot-bud formation (Fig. 1b) is possible in Dendrobium from different explants viz. leaf, nodal, flower stalk, root, rhizome and pseudobulb segments, thin-sections of shoot tips, leaves, protocorms and roots (Table 1). A PLB broadly describes an organ that develops in the in vitro culture of orchids that has a similar morphology, structure and function, as an enlarged seed-derived zygotic embryo, the protocorm (Arditti 1979; Teixeira da Silva 2014). Even shoot-bud or PLB formation from cell suspension cultures as well as scale-up of production through bioreactors (Fig. 1c) of several major commercial orchids has benefited Dendrobium micropropagation and transgenic research (Teixeira da Silva et al. 2011). Flowering must be controlled to meet market demands (Sim et al. 2007; Hsiao et al. 2011) and the effectiveness and reproducibility of the in vitro protocol is the first important step in ensuring the production of clonally similar germplasm for coordinated flowering in the greenhouse following acclimatization. The first such study for orchids has been done on somatic embryogenesis-specific marker, DcNAC (Dendrobium candidum NAC-like gene), which is expressed exclusively in the shoot apical meristem of PLBs (Zhao et al. 2011). This may serve as a new way to verify the existence of somatic embryogenesis in Dendrobium in vitro tissue cultures.

Fig. 1
figure 1

In vitro propagation of Dendrobium hybrids. Induction of somatic embryogenesis or protocorm-like bodies (a), multiple shoot induction (b), multiplication in a 5-L bioreactor system (c) and a chlorophyll mutant (d) (frequency in D. Visa Peach = 1/5,000, or 0.02 %) after 10 subcultures of Dendrobium shoot buds. Unpublished photos (Jean-Carlos Cardoso)

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Table 1 Micropropagation of Dendrobium species
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The main objective of this review is to provide a thorough understanding of the response of Dendrobium germplasm to in vitro conditions. By compiling what is known from a wide range of germplasm, literature and research laboratories around the globe, it is possible to establish a new biotechnological programme for a desired ornamental or medicinal Dendrobium species. Given the importance of members of this genus, not only for their ornamental value, but also for their medicinal value, an in vitro scale-up protocol using micropropagation of clonal material, has far-reaching economic implications. Moreover, also in the Dendrobium genus, other poorly studied and used aspects of in vitro clonally propagated material, such as efforts and possibilities for polyploidization, production of bioactive compounds, isolation of mutants and somaclonal variants, are discussed. Furthermore, in-depth knowledge of the response of Dendrobium in vitro would allow for related systems such as synthetic seeds, bioreactor technology, molecular genetics (Teixeira da Silva et al. 2014a), in vitro flowering (Teixeira da Silva et al. 2014c) and genetic transformation (Teixeira da Silva et al. unpublished review) to be achieved more successfully. Finally, an important objective of this review is to provide detailed insight into protocols that would allow for the preservation of currently endangered germplasm, or the application to closely related germplasm (Teixeira da Silva et al. 2014d).

Choice of explant, medium and constituents

There are a large number of studies dedicated to the tissue culture and micropropagation of Dendrobium from around the world. These 87 representative studies include 65 papers on 39 Dendrobium species and 22 papers on 20 Dendrobium hybrids (Table 1; Fig. 2). Most research focused on D. candidum (9 references including D. officinale which is frequently treated synonymously with D. candidum) (10.3 %), D. nobile and D. huoshanense (5.7 % for each species). The choice of explant is usually the most important initial factor that is considered when establishing a micropropagation protocol for orchids (Chugh et al. 2009), and may vary depending on the availability of material (if material is rare or endangered, then researchers may select to test several tissues or organs), seasonality of development (e.g., floral tissues only available during the flowering season), or level of infection and/or abundance of tissue (for example, in vitro shoot tips, leaves or nodal explants that are in a more juvenile and receptive state, would most likely be more suitable for initial culture establishment than mature, potted plants from a greenhouse). The explants most commonly used in micropropagation, derived from greenhouse-grown or in vitro Dendrobium plants, include nodal segments (23.0 %), in vitro-derived PLBs (21.8 %), shoot tips (11.5 %), protocorms (8.0 %), transverse thin cell layers (tTCLs) from protocorms and young stems (8.0 %), leaves (5.7 %), pseudobulbs (4.6 %), in vitro seedlings (4.6 %), axillary buds (3.4 %), and callus (1.1 %) (Table 1; Fig. 3), the in vitro-derived material not requiring a disinfection step. Aseptic young shoot buds (5–10 cm) are frequently used in Dendrobium micropropagation, from which lateral buds and the shoot tip are excised and inoculated in vitro. Axillary buds are also important for Dendrobium micropropagation as they are numerous and regenerate easily (JC Cardoso, unpublished data). Although solid, agarized medium is the preferred base, avoiding issues such as polyploidy and hyperhydricity, it is not uncommon to also observe liquid cultures at one or more stages of the in vitro protocol, or as the entire protocol. For example, Nogroho (2006) micropropagated D. ‘Emma Pink’ using liquid Vacin and Went medium (VW; Vacin and Went 1949) with constant agitation (90 rpm) and observed that formation of PLBs occurred in the second (56.3 % with PLBs) and third (43.8 % with PLBs) axillary buds, from the center of the young stem but that apical shoots could not regenerate PLBs. Liquid culture was essential for the success of bioreactor culture in D. ‘Zahra FR 62’ (Winarto et al. 2013).

Fig. 2
figure 2

Relative frequency (%) of micropropagation studies employing different Dendrobium species, cultivars or hybrids. All percentage values rounded down to one decimal place

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Fig. 3
figure 3

Relative frequency (%) of Dendrobium micropropagation studies employing different explants. PLB protocorm-like body, tTCL transverse thin cell layer. All percentage values rounded down to one decimal place. Total percentage values exceed 100 % since some studies employed more than one explant

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Axenic tissues, which are derived from pre-germinated seedlings, are frequently used to induce PLBs, which are in turn used to regenerate plantlets. For example, highest induction of PLBs (7.5/explant) in single pseudobulb segments sectioned from D. transparens seedlings germinated in vitro was obtained on ½ MS (half-strength, micro and macronutrients of the original Murashige and Skoog (1962) recipe) culture medium with 2.0 mg/l of 6-benzyladenine (BA) (Sunitibala and Kishor 2009). Ferreira et al. (2006a) used a modified liquid VW medium with Fe and micronutrients from MS culture medium supplemented with 2 % sucrose, 0.4 mg/l thiamine and 0.1 g/l myo-inositol, followed by the same culture medium supplemented with 0.45 µmol thidiazuron (TDZ) and 0.2 % Gelrite, to micropropagate D. ‘Second Love’ (Nobile group). These authors observed a positive correlation between the number of shoots/explant (1.06–6.24) and shoot dry matter (0.042–0.152 g) when the concentration of TDZ in culture medium was increased from 0 to 1.8 µmol. Kabir et al. (2013) found that Phytamax™ medium (Sigma Chemical Co., USA) was the most favorable for germination of D. fimbriatum seeds, with 100 % germination and the shortest germination period (45.5 days) compared to MS (93 %; 49 days) or VW (86 %; 49 days) media.

The basal media for the micropropagation of Dendrobium have included a wide range: MS (67 %), ½ MS, 2× MS, KC (Knudson 1946), ½ KC, VW, B5 (Gamborg et al. 1968), N6 (Chu et al. 1975), Phytotechnology medium, Mitra, modified RM medium (Kukulczanka and Wojciechowska 1983), these being the primary ones. Among these, MS, ½ MS, KC, VW were most often used (Fig. 4a) although their choice depended strongly on the explant employed (Fig. 4b).

Fig. 4
figure 4

Relative frequency (%) of Dendrobium micropropagation studies employing the five most common basal media (a) and the use of these media for different explants (b). B5 medium, or Gamborg medium (Gamborg et al. 1968); KC, Knudson C medium (Knudson 1921); Mitra, Mitra et al. (1976) medium; MS, Murashige and Skoog (1962) medium; VW, Vacin and Went (1949) medium. Values in parentheses in b indicate the absolute number of studies. All percentage values rounded down to one decimal place. Total percentage values exceed 100 % since some studies employed more than one basal medium

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A cytokinin (BA, kinetin (Kin), TDZ) and an auxin [indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA)] were usually used simultaneously in media (Fig. 5). Other cytokinins [N 6-isopentenyladenine (2-iP), zeatin (Zea)] and an auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), were also used for Dendrobium micropropagation (Das et al. 2008; Luo et al. 2009). The most commonly used plant growth regulators (PGRs) in Dendrobium micropropagation are BA (69.0 %) and NAA (65.5 %), used either as combined (56.9 %), or singly as BA (29.3 %) or NAA (13.8 %) in the culture medium (Fig. 5). Culture medium with no PGRs was also used in 17.2 % of studies (Fig. 5). Different PGRs have different effects on micropropagation of Dendrobium species (Table 1). The optimal medium is, as observed for many other plant species, including orchids (Hossain et al. 2013), dependent on the germplasm. For example, in one of the clearest studies demonstrating this dependence, Li et al. (2013c) reported the most suitable media for shoot and root induction among different species to be different. The suitable media for shoot induction were ½ MS + 0.5 mg/l BA + 0.1 mg/l NAA + 100 ml/l coconut water (CW) for D. pendulum and D. primulinum, and ½ MS + 0.25 mg/l BA + 0.1 mg/l NAA + 100 ml/l CW for D. heterocarpum. The suitable media for root induction were ½ MS + 0.5 mg/l NAA + 100 g/l banana pulp (BP) for D. pendulum and D. heterocarpum, and ½ MS + 0.75 mg/l NAA + 100 g/l potato pulp for D. primulinum. Semi-solid PGR-free ½ MS with 1 or 2 % sucrose was the best culture medium for increasing the growth of D. ‘Sonia-28’ PLBs (14.45 % increase) compared to liquid media containing full-strength or double-strength MS, or with BA and NAA in different combinations and concentrations. The main additives included in media are CW, AC, banana extract, peptone, among others (Fig. 6). Most papers used culture medium with no additives (58.6 %) and the main additive used in micropropagation of Dendrobium is CW (27.6 %). Those studies that employed CW, usually at a concentration of 10–25 % (v/v), employed no PGRs (41.7 %), included BA and/or NAA (45.8 %) or other PGRs or PGR combinations (12.5 %) (Fig. 6). The impact of CW on Dendrobium micropropagation may be caused by different biochemicals present in CW, including amino acids, vitamins, sugar, minerals and phytohormones (Yong et al. 2009). Peptone may induce changes in Dendrobium in vitro cultures since it generally consists of low molecular weight proteins, amino acids, vitamins and plant growth substances, which are able to enhance plant growth by providing plant cells with a readily available source of nitrogen (George et al. 2008). A tenable explanation for the positive effect of AC on Dendrobium micropropagation is that AC improves aeration and, at the same time, should microelements be added, establishes polarity, affects substrate temperature, or absorbs toxic substances, including phenolics (Zeng et al. 2015a, b).

Fig. 5
figure 5

Relative frequency (%) of Dendrobium micropropagation studies employing different plant growth regulators. 2,4-D 2,4-dichlorophenoxyacetic acid, BA N6-benzyladenine, IAA indole-3-acetic acid, IBA indole-3-butyric acid, Kin kinetin, NAA α-naphthaleneacetic acid, PGR plant growth regulator, Put putrescine, Spd spermidine, Spm spermine, TDZ thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea), TRIA triacontanol. All percentage values rounded down to one decimal place. Total percentage values exceed 100 % since some studies employed more than one PGR

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Fig. 6
figure 6

Relative frequency (%) of Dendrobium micropropagation studies employing different additives. AC activated charcoal, CW coconut water. All percentage values rounded down to one decimal place. Total percentage values exceed 100 % since some studies employed more than one additive

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The main gelling agents used are agar (49.3 %), gelrite and phytagel (19.3 %) (Fig. 7), while the most common pH of the medium was 5.8 although a large percentage of studies (33.3 %) did not report the pH of the medium (Fig. 8).

Fig. 7
figure 7

Relative frequency (%) of Dendrobium micropropagation studies employing different gelling agents. NR not reported. All percentage values rounded down to one decimal place. Total percentage values exceed 100 % since some studies employed more than one gelling agent

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Fig. 8
figure 8

Relative frequency (%) of Dendrobium micropropagation studies employing different pHs in the culture medium. NR not reported. All percentage values rounded down to one decimal place (thus total is less than 100 %)

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Culture temperature and light

In most studies on Dendrobium micropropagation, the temperature ranged from 22 to 29 °C, but 25 ± 2 °C (57.5 %) was most commonly used in 50 studies. The photoperiod was 10–16 h and illumination intensity was 350–3,000 lux or 13.5–150 µmol m−2 s−1, the 16-h PP (42 %) although 1,000–2,000 lux (20.5 %) or 30–60 µmol m−2 s−1 (43.2 %) was most commonly used, with only 6.82 % of reports using dark culture and 1.14 % employing light-emitting diodes (Tables 1, 2).

Table 2 Meta-analysis of the light intensity employed in Dendrobium micropropagation studies (all values in relative frequency (%))
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Thin cell layers, somatic embryogenesis, and protoplasts

The thin cell layer (TCL) culture system, which is a promising and efficient technique with regard to the total output of plantlets than conventional in vitro methods and explants for the in vitro regeneration of orchids (Teixeira da Silva 2013a; Teixeira da Silva and Dobránszki 2013), has also been employed in select studies for the in vitro propagation of Dendrobium (Nayak et al. 2002; Malabadi et al. 2005; Rangsayatorn 2009; Jaiphet and Rangsayatorn 2010; Table 1). Kaewubon et al. (2014) attributed the browning of callus originating from tTCL protocorms of D. crumenatum Sw. to disorganizations at the cellular level, such as thickened and distorted cell wall, lack of cytoplasm, fewer plastids, the presence of many small vehicles and cellular decompartmentation, the presence of phenolic compounds and decreased carbohydrate accumulation. Khosravi et al. (2008) credited callus browning of Dendrobium cv. ‘Serdang Beauty’ to ethylene production due to inadequate PGR supply in the medium. The advantage of the TCL system for Dendrobium is the ability to produce a high frequency of shoot regeneration or to enhance PLB formation while reducing the time interval is required to achieve organogenesis. Using the Plant Growth Correction Factor, it would now be easier to make direct comparisons between protocols that involve different explant sizes, including TCLs and other explants (Teixeira da Silva and Dobránszki 2011, 2014).

Somatic embryogenesis (involving somatic embryos of single-cell origin) is another potential method to micropropagate orchids. PLBs are in fact equivalent to somatic embryos (Teixeira da Silva and Tanaka, 2006), making synseed technology, low temperature storage and cryostorage more realistic, and possible (Teixeira da Silva 2012a, b; Bustam et al. 2013; Sharma et al. 2013; Antony et al. 2014). Spontaneous formation of organs that resemble protocorms, the PLBs—used specifically for orchids—are achieved in two ways, either through direct embryogenesis i.e. PLBs develop without intervening callus phase, or through indirect embryogenesis, i.e. PLBs develop with an intermediary callus phase (Hossain et al. 2013; Teixeira da Silva 2013b; Table 1; Figs. 1, 9a–c). Since PLBs and somatic embryos are synonymous in orchid biotechnology, the terms somatic embryogenesis and PLB formation, refer to the same developmental event. The combinations, concentrations, and the ratio of plant growth regulators (PGRs) are critically important, as is the choice of basal medium, light and other abiotic conditions and medium additives. These aspects are detailed for dozens of Dendrobium species and cultivars in Table 1. These systems would facilitate commercial micropropagation of orchids because they have very high regeneration potential compared to other methods and because embryogenic callus is an appropriate target material for genetic transformation (Teixeira da Silva et al. 2011). TDZ may currently be the most potent and useful PGR for PLB regeneration from different tissues of Dendrobium species and hybrids, as observed by different authors (Table 1; Chung et al. 2005; Roy et al. 2007; Sujjaritthurakarn and Kanchanapoom 2011).

Fig. 9
figure 9

In vitro development of Dendrobium officinale Kimura & Migo. a Mixture including callus, protocorm-like bodies (PLB) and shoots on N6 medium containing 2.0 mg/l N 6-benzylaminopurine (BA) and 10 % coconut water (CW) from nodal segments explants ex vitro; b proliferation and differentiation of callus, PLBs and shoots on N6 medium containing 1.0 mg/l BA and 10 % CW; c plantlets regenerated from PLBs on N6 medium containing 1.0 mg/l α-naphthaleneacetic acid (NAA); d rooting and seedling growth on N6 medium containing 1.0 mg/l NAA, 100 g/1 banana homogenate and 1.5 g/l activated charcoal. Unpublished photos (Songjun Zeng)

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Protoplasts, a useful tool for plant regeneration, gene introduction and fusion experiments, have been isolated from Dendrobium seedlings (Teo and Neumann 1978) while cases of successful colony formation in Dendrobium are limited (Kuehnle and Nan 1990; Kunasakdakul and Smitamana 2003; Khentry et al. 2006; Tee et al. 2011). Isolation and culture conditions for Dendrobium Sonia ‘Bom 17’ protoplasts were optimized by Khentry et al. (2006). In that study, protoplasts were isolated from the leaves of in vitro plantlets with a yield of 3.97 × 105 protoplasts/g fresh weight by digesting with an enzyme solution of 1 % Cellulase Onozuka R-10, 0.2 % Macerozyme, 0.3 M mannitol, 10 mM CaCl2·2H2O and 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES) for 4 h. After washing in 0.3 M mannitol solution with 10 mM CaCl2·2H2O and 10 mM MES, and purifying in 0.3 M sucrose, protoplasts (2 × 105 protoplasts/ml) were cultured in an agarose-bead culture on Kao-Michayluk medium (Kao and Michayluk 1975) in the dark at 25 °C. In two-day-old cultures, the first cell divisions occurred, and after 2 weeks, multicellular colonies with 15–20 cells formed. However, no further proliferation occurred after 3 weeks therefore no callus proliferation was observed. Protoplasts from mesophyll cells of D. ‘Pompadour’ could be isolated by digesting with an enzyme solution containing 1 % Cellulase Onozuka R-10, 1 % Macerozyme R-10, 0.5 % Driselase, and 0.4 M mannitol for 3 h on a gyratory shaker (80 rpm). After purification of protoplasts on a sucrose gradient, they were fused using 40 % polyethylene glycol. As the osmotic gradient was lowered, fusion occurred (Kanchanapoom et al. 2001). A combination of 2 % cellulose, 2 % pectinase and 0.5 M sorbitol applied for 4 h was ideal for protoplast isolation from in vitro leaves of D. crumenatum yielding a density of 28.66 × 104 protoplasts/g fresh weight (Tee et al. 2010). No new studies between 2011 and 2014 related to protoplasts have been published but protoplasts remain an important biotechnological tool for inter-taxon breeding.

Rarely studied aspects of Dendrobium in vitro culture

Only a single case of ultrasound in the improvement of Dendrobium in vitro cultures documents how the conversion of PLBs to shoots can be improved by applying 300 W of ultrasound for 5 min (Wei et al. 2012).

Colchicine has been used to convert diploid to tetraploid forms in Dendrobium (Chaicharoen and Saejew 1981; Sanguthai et al. 1973; Li et al. 2004; Atichart and Bunnag 2007) while polyploids were induced by Miguel and Leonhardt (2011) with oryzalin. Also, ex vitro induction of tetraploids was possible in D. nobile using a solution of 0.1 % colchicine for 96 h to obtain 29.17 % of autotetraploid plants (Vichiato et al. 2007). Ploidy analysis using flow cytometry in Dendrobium is possible (Jones and Kuehnle 1998; Atichart and Bunnag 2007; Seah 2009; Zhang et al. 2009; Teixeira da Silva et al. 2014b), as well as by traditional chromosome counting (Vichiato et al. 2007). Understanding changes in ploidy serves as one technique to assess how a tissue culture technique may influence the stability of cultures.

Although large-scale commercialization of photoautotrophic micropropagation has not been achieved for Dendrobium yet, it can serve as an alternative to heterotrophic or photomixotrophic micropropagation in the future for high-scale in vitro propagation of plants (Xiao et al. 2011). One pilot study exists in which D. candidum was successfully micropropagated photoautotrophically by controlling vessel ventilation, light intensity, and CO2 concentration (Xiao et al. 2007).

Bioreactors can counter the weaknesses of micropropagation and can benefit orchid propagation by easily producing a large number of plantlets, scaling up production, easy handling of inoculation or harvesting of cultures, and saving labor cost and time. Bioreactors had only been applied for the propagation of a limited number of orchid genera such as Phalaenopsis, Anoectochilus and Oncidium (Hossain et al. 2013), but Dendrobium can also be effectively mass propagated by bioreactor culture, as demonstrated in 3-l (Winarto et al. 2013) or 5-l (Fig. 1c) bubble reactors. Production of bioactive compounds is another aspect of using bioreactors. A balloon-type bubble bioreactor was used to produce polysaccharides, coumarins, polyphenolics, flavonoids and vitamin E and C using a protocorm suspension culture from D. candidum Wall ex Lindl (Cui et al. 2014).

The identification of mutants in micropropagated plantlets using molecular markers is an important tool in orchid micropropagation. By identifying mutants or somaclonal variation in the in vitro step will avoid possible negative repercussions such as delayed flowering, mutant colors, non-standard timing of flowering, or non-uniform height or architecture of potted plants, which often take a few years to achieve maturity after acclimatization. Even though molecular markers have been quite extensively used for other horticultural crops to assess variation of in vitro cultures, their use in Dendrobium micropropagation is still quite limited, and has only employed a single type of molecular marker. Ferreira et al. (2006b) observed 172 fragments with 5–12 distinct and reproducible bands per primer using rapid amplified polymorphic DNA (RAPD) molecular markers and did not observe alterations in DNA in micropropagated D. ‘Second Love’ plantlets. Liu et al. (2005) found no variation among in vitro D. officinale seedlings when subcultured 1–3 times, but some small variation among seedlings of the 4th–7th subcultures was detected using RAPD molecular markers. Since many Dendrobium species are important medicinal plants, molecular markers are an absolute necessity to avoid errors and to prevent adulteration.

Conclusions and future perspectives

The micropropagation of Dendrobium and the conditions that define the optimal parameters for its tissue culture are essential for subsequent success of acclimatization and uniform growth and flowering in the greenhouse. This review has identified the most commonly used parameters in the tissue culture of Dendrobium species, hybrids and cultivars: use nodes or PLBs as the explant source, MS as the basal medium, BA and NAA in combination, no additives or the use of CW, agar as the gelling agent at pH 5.8, and a low light intensity (20–60 µmol m−2 s−1). These conditions have resulted in the reported optimized protocols for a wide range of germplasm, but the reader is cautioned that the combination of these most frequently reported conditions might not collectively represent the ideal protocol for any, or all, germplasm represented. The objective of optimizing in vitro parameters serves to not only reproduce protocols reported in the literature, and apply them to the commercial production at a larger scale, but also serves as a stable system to establish uniform cultures that would allow for more applied biotechnological applications such as genetic transformation, phylogenetic studies, in vitro flowering, or developmental studies. This review provides the most comprehensive assessment of the Dendrobium in vitro literature to date, summarizing the optimal conditions as defined by different authors, for a wide range of hybrids and species. This compilation will allow Dendrobium research to advance more rapidly since many aspects, basic and applied, have yet to be studied.

Author contributions statement

All four authors contributed equally to all aspects related to the review.