The importance of medicinal and ornamental Dendrobium

The genus Dendrobium s.l. (Epidendroideae) has in excess of 1100 species of epiphytic orchids with a wide distribution from Central Asia throughout Australasia (Kamemoto et al. 1999; Kumar et al. 2011). This genus is one of the largest among the Orchidaceae, the largest family of angiosperms (Dressler 2005; Fay and Chase 2009). Species within the Dendrobium genus are highly prized ornamental assets, primarily as potted plants with showy flowers (Fig. 1a) that tend to have a long vase life (Vendrame et al. 2008). But the most important aspect of many orchid species, including Dendrobium species, is their medicinal and pharmaceutical value, particularly Dendrobium nobile Lindl., which is abundantly used in traditional Chinese medicine (Bulpitt et al. 2007; Singh and Duggal 2009; Ng et al. 2012).

Fig. 1
figure 1

In vitro development of Dendrobium officinale Kimura and Migo. a Flowering plant; b asymbiotic seed germination on N6 solid medium containing 10 % coconut water (CW), 0.2 mg/l α-naphthaleneacetic acid (NAA), 1 g/l activated charcoal (AC), 0.65 g/l agar powder; c asymbiotic seed germination on N6 liquid medium containing 0.2 mg/l NAA, 10 % CW. Unpublished photos (Songjun Zeng)

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To counter exploitation from wild resources, and to bolster production of clonal material, biotechnology—specifically micropropagation (Teixeira da Silva et al. 2015a), in vitro flowering (Teixeira da Silva et al. 2014a), cryopreservation and low-temperature preservation (Teixeira da Silva et al. 2014b)—serves as an important tool for propagation and preservation purposes (Roberts and Dixon 2008; Swarts and Dixon 2009). They also allow for the establishment of sterile in vitro cultures for the study of the genetics of flowering in orchids (Teixeira da Silva et al. 2014c). Another strategy is to use seeds aseptically germinated in vitro, under optimized conditions, modeling natural ones. The ease and high germination percentage of seeds of Dendrobium species (Table 1) under asymbiotic conditions relative to the more precise requirements of symbiotic germination, which needs to balance germination and pathogenesis (Teixeira da Silva et al. 2015b), makes this technique more practical and useful for mass in vitro propagation and/or germplasm conservation (Teixeira da Silva et al. 2014b).

Table 1 Asymbiotic seed germination in vitro of Dendrobium species (listed chronologically, then alphabetically within each year)
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Although many commercial Dendrobium hybrids are propagated using clonal procedures, asymbiotic seed propagation in Dendrobium has major importance for the conservation and propagation of wild species because of loss of habitats and overexploitation due to agriculture, urbanization, overcollection and medicinal uses. Dendrobium orchids are commonly used in traditional Chinese medicine and many wild populations, for example, of D. catenatum Lindl., have become drastically reduced due to overexploitation (Liu et al. 2011; Luo et al. 2013). The propagation of rare and wild species using asymbiotic germination for economic ornamental, as well as for conservation/restoration purposes, is another important market for Dendrobium, mainly because each mature capsule contains 2–3 million seeds with a high percentage of viable seeds that can germinate under in vitro asymbiotic conditions (Paul et al. 2012). Another commercially important application of asymbiotic germination is in breeding programmes aimed at accelerating the speed at which new cultivars are obtained (Cardoso 2012).

This review aims to examine the available literature (53 studies; Table 1) related to asymbiotic in vitro Dendrobium seed germination (covering 37 species and multiple hybrids; Table 2) in a bid to identify those trends that could assist researchers better formulate a study or conservation programme for commercial or for rare species and hybrids. All nomenclature has been verified on The Plant List (http://www.theplantlist.org/).

Table 2 Species and seed stages or ages used for in vitro asymbiotic germination of Dendrobium species
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Seed germination in vitro

Seed germination is defined as the emergence of radicle through the seed coat and involves a sequence of previous steps, as imbibition of water followed by embryo expansion (Figs. 1, 2a, b), resulting in initial seedling development under favorable conditions (Manz et al. 2005). Nowadays, germination of orchid seeds is easy, though a labor-intensive procedure (Kauth et al. 2008), but several advances have taken place, as outlined in Table 1. The first investigators, L. Knudson and N. Bernard, reported difficulty in germinating orchid seeds due to environmental conditions and small size of embryos (Knudson 1922) and, as described by Bernard (1909), strong dependence on mycorrhizal fungi that inhabit the roots of orchids. Knudson (1922) was able to germinate an epiphytic orchid (Laelia–Cattleya hybrid) in aseptic mineral media supplemented with diverse types of carbohydrates such as glucose and sucrose.

Fig. 2
figure 2

Imbibed seeds on solidified agar medium (pH 5.7), without sugar, nutrients and additives in light (16-h photoperiod) (a). Swollen seeds of Dendrobium aqueum on basal medium with half-strength MS macronutrients in the dark after 4 months (b). Unpublished photos (Parthibhan et al.)

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Pod or seed sterilization

The sterilization of Dendrobium pods, as recorded in the literature (Table 3), shows several patterns and trends (Teixeira da Silva et al. 2015b). The sterilization of undehisced Dendrobium pods, having immature and mature seeds, starts with a wash in running tap water containing Teepol, a detergent (1–20 %; 20 min) or a household or commercial bleach (12 %; 20 min). Surface-disinfected pods are first transferred to a laminar air hood and surface sterilized with ethanol (EtOH; rubbed or dipped, 70–90 %; 20–30 s or 1–15 min). Some Dendrobium pods (D. aphyllum (Roxb.) C.E.C.Fisch., D. aqueum Lindl., D. bigibbum Lindl. var. compactum, D. chrysanthum Wall. ex Lindl., D. chrysotoxum Lindl., D. formosum Roxb. ex Lindl., D. hookerianum Lindl., D. longicornu Lindl., D. nobile, D. parishii Rchb.f.) are also flamed (Dohling et al. 2008; Hajong et al. 2010; Kananont et al. 2010; Vasudevan and Van Staden 2010; Dutta et al. 2011; Kaewduangta and Reamkatog 2011; Parthibhan et al. 2012; Paul et al. 2012; Hossain 2013; Nongdam and Tikendra 2014). Additional treatment, either alone or in combination, involves HgCl2 (0.1–1.0 %; 8–12 min) and NaOCl (1.0–10 %; 5–15 min), which were also reported to be effective. The reported type, concentration, and duration of exposure of sterilants differ considerably, and thus need to be standardized for any species. EtOH and HgCl2 are most frequently used for seed sterilization, and have a protein-denaturing property that exterminates bacteria while NaOCl (the second most used sterilant for seeds), an alkali, loses chloride causing the active oxidizing ion to capture oxygen, thus killing aerobic microorganisms and fungal spores that are mainly responsible for most of the contaminations (Alvarez-Pardo et al. 2006). EtOH is usually used in asymbiotic seed germination with an additional source of chlorine for complete sterilization, usually derived from HgCl2 (in 28.3 % of papers) or NaOCl (in 15.1 % of papers). EtOH followed by flaming is also applied for the sterilization of mature and green capsules (Tables 1, 3; Teixeira da Silva et al. 2015b). In some instances, KMnO4, a commercial fungicide (Bavistin), a bactericide (streptomycin sulfate), and even sonication were also applied for better sterilization. To start Dendrobium in vitro culture, mature seeds and pods are the main explants (30.2 and 24.5 %, respectively) (Table 3).

Table 3 Sterilization agents used for different types of organs used in papers related to in vitro asymbiotic seed germination of Dendrobium species
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Media composition

Orchid plants produce numerous minute seeds and each seed contains insufficient nutrient reserves for germination. The establishment of a symbiosis with an appropriate fungus is indispensable for germination under natural conditions. In vitro, orchid seed germination can be achieved using either asymbiotic or symbiotic methods (Rasmussen 1995; Yam and Arditti 2009; Fig. 1), which are strongly influenced by several abiotic factors, including medium composition and culture conditions. This is likely a genotype-specific response, as was observed in Cypripedium spp. (Zeng et al. 2015). MS and ½ MS are the most commonly used basal culture medium for Dendrobium seed germination, while KC or modified KC and N6 are the second most used formulations (Fig. 3).

Fig. 3
figure 3

The percentage of culture media used in papers on asymbiotic germination of Dendrobium. Campos modified (Soares et al. 2013), KC Knudson’s C medium (Knudson 1921), MS Murashige and Skoog (1962) medium, N 6 medium Chu’s medium (Chu et al. 1975), PT medium (Zeng et al. 1998), VW Vacin and Went (1949) medium. The sum of percentages may exceed 100 % because some authors used or recommended more than one culture medium in the same paper. In the case that authors used several culture media, only the best media were used to calculate the percentage values

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OMF

Several studies on in vitro symbiotic Dendrobium seed germination with cultures of OMF (Swangmaneecharern et al. 2012; Wu et al. 2012) and asymbiotic germination using liquid extract of OMF (Guo and Xu 1990) support the favorable role of mycorrhizae (Teixeira da Silva et al. 2015b). Swangmaneecharern et al. (2012) reported the effectiveness of five isolates of the OMF Epulorhiza sp. (isolated from Paphiopedilum, Dendrobium, and Cymbidium) in promoting seed germination and protocorm development of four Dendrobium species (D. pulchellum Roxb. ex Lindl., D. crepidatum Lindl. and Paxton, D. findlayanum E.C. Parish and Rchb. f. and D. crystallinum Rchb. f.). However, the promoting effects of different fungal isolates on seed germination of each orchid species were not equal: the isolates Da-KP-0-1 (from Dendrobium) and Ps-KT-0-1 (from Paphiopedilum) were effective in D. pulchellum and D. crepidatum, whereas the isolates Pv-PC-1-1 (from Paphiopedilum) and Da-KP-0-1 (from Dendrobium) were reported to be the best for D. findlayanum.

Basal media

Although PGR-free basal media usually (54.7 % of papers) support the in vitro germination of Dendrobium seeds (Fig. 4), and have been used for many orchids (Parthibhan et al. 2012; Paul et al. 2012; Hossain 2013; Teixeira da Silva 2013), several PGRs have also been effectively used. The most frequently used PGRs for the asymbiotic germination of Dendrobium seeds are α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), or a combination of both (Fig. 4). Even though in most studies (50.9 %) no special additives were used (Fig. 5), diverse organic compounds (malt, yeast, casein, peptone, beef and tryptone extracts at 0.01 to 0.1 g/l) and natural supplements (banana pulp, coconut water (CW), potato juice, sugarcane juice and tomato juice at 5 to 15 %) can greatly affect (improve or inhibit) Dendrobium germination (Fig. 4; Teixeira da Silva 2013; Parthibhan et al. unpublished). CW is the main natural supplement used for this aim (13.2 % of papers) (Fig. 5). Orchid seeds prefer and require external nutrients or growth substances for effective germination and/or seedling growth in nature and in vitro. Unlike epiphytic orchids, terrestrial orchid seeds will not grow beyond the protocorm stage unless infected by a suitable mycorrhizal fungus, since they are subterranean in nature. Even after a suitable symbiotic relationship has been established, non-green protocorms can take many weeks, months or even years to grow and produce leaves and roots, depending upon the species, to maintain a mutualistic symbiosis (McKendrick 2000). Thus, in general, as a result of this relationship, terrestrial orchids only require a low concentration of nutrients or more diluted media such as ½ MS, ¼ MS and MS for some Paphiopedilum species (Hossain et al. 2013a; Teixeira da Silva 2013; Zeng et al. 2012, 2014, 2015), when germinated symbiotically in vitro, because of mycotrophy (Rasmussen 1995). Epiphytes prefer high nutrition or concentrated media and a carbon source for germination and seedling development (Arditti 1979). Despite this, highly viable D. aqueum seeds remain ungerminated even after imbibition for 5 months on nutrient- and sugar-free solidified agar (0.7 %) medium (Parthibhan et al. unpublished).

Fig. 4
figure 4

The percentage of studies that used plant growth regulators, alone or in combination, in basal culture media for asymbiotic germination of Dendrobium species. BA N6-benzyladenine (BA is used throughout even though BAP (6-benzylamino purine) may have been used in the original, according to Teixeira da Silva (2012), GA 3 gibberellic acid, IAA indole-3-acetic acid, Kin kinetin, NAA α-naphthaleneacetic acid, PGR plant growth regulator

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Fig. 5
figure 5

The percentage of studies on asymbiotic germination of Dendrobium species that used additives, either alone, or in combination. AC activated charcoal, BE banana extract, BH banana homogenate, BP banana pulp, CW coconut water, NR not reported, PH potato homogenate

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McKendrick (2000) suggested that when attempting to germinate a new species, it is important to test media at both full and half strength to determine the best nutrient base. Since Dendrobium species and hybrids have commercial, ornamental and medicinal value, conservation strategies are also required (Bulpitt 2005; Bulpitt et al. 2007; Kuehnle 2007; Teixeira da Silva et al. 2014b). A combination of asymbiotic seed germination and vegetative propagation forms the basis of economic horticultural production of orchid plants (Smith and Read 2008).

Asymbiotic seed germination is used to accelerate and increase the efficiency of germination of Dendrobium orchids. However, the relatively ease with which they can be micropropagated using clonal techniques, such as shoot tip or axillary bud culture, rather than seed propagation, presents several advantages for commercial production, especially for the flower market. Despite this, in many countries, most micropropagation research was developed using seeds as initial explants (Table 1). Orchids, including the genus Dendrobium, present high heterozygosity and seed propagation results in high genetic variation of progeny (Gu et al. 2007; Chattopadhyay et al. 2012). Faria et al. (2004a) observed significant variation in morphological characteristics, such as height (2.38 to 6.10 cm), number of roots (2.8 to 8.7), fresh (0.41 to 1.95 g) and dry (0.03 to 0.12 g) weight from 20 different crosses and self-pollination obtained from different cultivars of in vitro cultivated D. nobile plantlets. Lone et al. (2008) also observed a variation of 4.23–6.89 cm in plant height, 5.2–12.87 roots/plantlet, and 0.23–0.63 g of total fresh weight/plantlet after 109 crosses using D. phalaenopsis. Seed propagation is commonly used for conservation of endangered species, and for breeding purposes (Faria et al. 2004b; Lone et al. 2008; Cardoso 2012). In these cases, genetic variation is a positive factor expected for conservation or development of new cultivars.

A large number of media types were tested in Dendrobium seed germination and seedling development (Table 1). These media include MS, half- or a quarter-strength (the latter not being recommended) of MS macro- and microelements (½ MS or ¼ MS, respectively), Chu’s N6 medium (Chu et al. 1975), B5 (Gamborg et al. 1968), Vacin and Went (1949; VW), KC, or Hyponex (Chen et al. 2004; Yang et al. 2006) media (Fig. 3). The ideal medium for germination of each Dendrobium species differs. KC or modified KC media are usually used for Dendrobium seed germination (Table 1; Hu and He 1979; Xu and Yu 1984; Yu et al. 2011). Parthibhan et al. (2012) studied 20 basal media devoid of PGRs and additives for D. aqueum. Half-strength MS medium macronutrients, to which 2 % sucrose was added, resulted in highest seed germination (93.41 %) and seedling development. Zeng et al. (1998) reported that the optimal medium for embryo culture of five Dendrobium species was N6, to which 0.2 mg/l NAA was added. Song et al. (2004) reported that D. nobile seed germinated better on modified KC medium than on VW medium. For seed germination, de Moraes et al. (2010) used 30 ml of MS medium with 7 g/l of agar and pH adjusted to 6.0. MS medium resulted in highest germination (90–95 % and 80–85 %) of D. longicornu and D. formosum, respectively (Dohling et al. 2008). Hajong et al. (2010) reported the best germination (94 %) on PGR-free MS medium in D. chrysanthum under a 12-h photoperiod. Similar results were obtained by Paul et al. (2012) with D. hookerianum in which maximum germination percentage (95.27 ± 0.68 %) was observed using MS medium, when compared with Mitra (87.85 ± 0.81 %), KC (73.00 ± 1.23 %) and B5 (51.38 ± 1.31 %) media. The same authors also observed more rapid germination (about 2 weeks after inoculation) and the best development of seedlings from protocorms on MS medium.

Carbohydrates

Sucrose has served as the main source of carbohydrate used for micropropagation of Dendrobium orchids, and only in 2.2 % of Dendrobium micropropagation studies have other carbohydrates improved the effect of sucrose (Teixeira da Silva at el. 2015a). Luo et al. (2009) tested the effect of sucrose, maltose, glucose and fructose at 5–40 g/l and found that the best shoot development of D. huoshanense from PLBs was achieved on medium with 10 g/l maltose. Glucose and fructose were best for PLB proliferation of D. ‘Alya Pink’ among six carbohydrates tested (mannitol, galactose, sorbitol, glucose, sucrose or fructose, all applied at 2 %) (Nambiar et al. 2012). Faria et al. (2004a) tested five sucrose concentrations (0, 5, 10, 20, 30, and 60 g/l) in ½ MS at pH 5.8 for in vitro cultivation of D. nobile seedlings. The best plant height (4.21 cm), fresh weight (0.17 g), root length (4.75 cm) and number of shoots (4.4) resulted after 120 days of cultivation using 60 g/l sucrose. Similarly, sucrose was the carbohydrate source when culture medium contained an artificial carbohydrate (21.2 % of the studies shown in Table 1) during asymbiotic seed germination of Dendrobium. The seed germination percentage of D. nobile hybrids was not modified by a sucrose concentration between 10 and 40 g/l in all stages (Udomdee et al. 2014; Table 1). However, as sucrose concentration increased, the number of seedlings with two or more leaves and roots (stage 6) decreased, and seedling development was most rapid, developing to stage 6, when 10 g/l sucrose was used.

Plant growth regulators

Seed germination of Dendrobium germplasm is usually enhanced by the inclusion of PGRs in medium. BA, kinetin (Kin), NAA, and GA3 have different effects on seed germination and seedling growth of different Dendrobium species (Mo and Ling 2007; Cui et al. 2012; Fu et al. 2012; Li et al. 2013). In D. transparens Lindl., 90 % germination was achieved on B5 medium supplemented with 1.0 mg/l Kin and 1.0 mg/l NAA (Hazarika and Sarma 1995). Soares et al. (2012) observed better germination of D. nobile in PGR-free medium (46.47–49.47 % germination): when BA or GA3 was added, germination percentage decreased to 1.68 and 9.06 %, respectively. The optimum medium composition for germination of D. candidum was VW basal medium containing 0.18 mg/l NAA, 0.53 mg/l BA, 0.28 mg/l GA3, 20 g/l sucrose, 100 g/l banana homogenate (BH) and 10 g/l AC (Du et al. 2007). D. chrysotoxum seeds germinated on Mitra medium supplemented with AC (0.4 %), BA (2 mg/l) and IAA (2 mg/l) showed enhanced germination (as much as 98.10 %) compared to PGR-free medium (82.4 %) (Nongdam and Tikendra 2014). Hossain (2013) noted that MS medium was more efficient for germination of D. aggregatum seeds than Phytamax™ (PM; Sigma Chemical Co., USA) medium and the addition of 2.0 mg/l BA and 1.0 mg/l NAA caused the profuse development of secondary protocorms from primary protocorms; the most effective medium for plantlet formation was MS containing 0.5 mg/l IAA. In contrast to these results, Kabir et al. (2013) noted that in 100 % of flasks, D. fimbriatum Hook. seeds germinated using PM culture medium (more effective than MS or modified VW), although further seedling elongation was achieved when liquid MS medium was supplemented with 2.0 mg/l BA and 0.01 mg/l IBA. Shoots, on the other hand, were induced in the presence of 1.0 mg/l BA and 0.5 mg/l picloram, while ½ MS supplemented with 1.0 mg/l IAA was most suitable for effective induction and growth of adventitious roots.

Organic and inorganic additives

Dendrobium seed germination and protocorm development are stimulated or inhibited by organic amendments including coconut water (CW; Fig. 2a, b), apple homogenate (AH), BH, or potato homogenate (PH) (Lo et al. 2004a; Zhan et al. 2010; Gong et al. 2015). Su et al. (2012) observed that a simple culture medium using 3 g/l of commercial fertilizer (8 % nitrogen, 9 % P2O5, 9 % K2O) and banana paste/pulp (BP) (6 % w/v) used for in vitro D. nobile cultivation resulted in taller plantlets (8.06 vs 6.65 cm in the control), but with fewer leaves (6.84 vs 7.10 in the control) and roots (8.86 vs 7.10 in the control), greater dry weight (0.81 vs 0.58 g in the control) and a longer main root (5.48 vs 4.22 cm in the control), when compared to ½ MS culture medium with BP (6 % w/v). Song et al. (1999) also observed that 6 % BP increased the in vitro growth of D. nobile plantlets. Ngampanya and Homlaaor (2010) also reported that the addition of BP and CW promoted seed germination of Dendrobium sp.

A popular complex mixture used for Dendrobium orchid cultivation in vitro is CW (Fig. 5). Soares et al. (2013) used a complex culture medium consisting of a mixture of 70 g/l tomato, 50 g/l BP, 3 ml of commercial fertilizer (containing 10 % N, 10 % P2O5, and 10 % K2O), 25 g/l commercial sugar, 3 g/l AC and 17 g/l of agar, at pH 5.0. To that medium, 0, 50, 100, 150 or 200 ml/l of CW was added. Highest number of pseudobulbs (2.5/plantlet), leaves (6.9/plantlet) and roots (5.6/plantlet), height of plantlets (2.2 cm), shoot fresh weight (0.5 g), and root length (5.4 cm) was observed when 20 % (v/v) of CW was used compared to the control (1.87/plantlet, 4.74/plantlet and 4.10/plantlet; 1.37 cm, 0.24 g and 4.57 cm). Half MS medium supplemented with 20 % potato extract resulted in highest germination of D. candidum (Tang et al. 2005). Zeng et al. (1998) reported 10 % CW (v/v) to be the best for seed germination of D. wangliangii G. W. Hu, C. L. Long and X. H. Jin, D. candidum, D. densiflorum Lindl., D. fimbriatum and D. loddigesii Rolfe. Similar conclusions were made by Soares et al. (2013) using 20 % CW (v/v) for D. nobile and by Vijayakumar et al. (2012) using 15 % CW (v/v) for D. aggregatum. After shoot emergence, adding potato juice, green-bean seedling juice or banana juice can improve the growth of plantlets, although banana juice was observed to be the best for rooting (Ye et al. 1988; Ding 2004; Lo et al. 2004b; Yang et al. 2006; Kong et al. 2007; Sun et al. 2009; Vyas et al. 2009; Qian et al. 2013). Lo et al. (2004a, b) reported that full-strength MS basal medium supplemented with 8 % BH, 8 % PH, or 8 % CW improved seedling growth of D. tosaense, D. moniliforme (L.) Sw., and D. linawianum Rchb.f. than when ½ MS medium with 3 % sucrose was used.

Kananont et al. (2010) reported that the responses of seed germination and protocorm formation to chitosan were dependent on the species and developmental stage. All six tested types of chitosan polymers or oligomers formed with 70, 80 or 90 % deacetylation (P70, P80, P90, O70, O80 and O90), and five concentrations (0, 10, 20, 40 and 80 mg/l) significantly enhanced the proportion of D. formosum seeds (90.6 and 91.2 %) that germinated compared with the control (67.8 %). In contrast, only 10 mg/l of O70 or P80 chitosan resulted in enhanced seed germination (15.0 % and 13.7 %) compared to the control (10.5 %). Further protocorm growth of D. bigibbum var. compactum was significantly improved on chitosan at 10 mg/l, except for O90, whilst 10 or 20 mg/l of P70 chitosan enhanced the growth of D. formosum protocorms the most.

Agar can be replaced in part by other types of gelling agents. Soares et al. (2014) observed that the use of 7 g/l agar + 7 g/l corn starch resulted in better development of D. nobile shoots and roots, resulting in a 188, 156 and 177 % increase in the number of leaves, roots and fresh weight, respectively, compared with 14 g/l of agar for control seedlings.

Results regarding the use of activated charcoal (AC) are contradictory. Galdiano-Júnior et al. (2011) used MS basal medium with half the concentration of macronutrients (½ MS), 2 % sucrose, 7 g/l agar and pH 5.7, and tested 0, 1 and 2 g/l of AC applied to the in vitro culture (from seed germination to the first subculture of seedlings) of D. nobile seedlings for 180 days. They observed that AC, independent of the concentration, reduced the number of roots (29.7 %), length of the main root (47.0 %), number of leaves (13.6 %), total fresh (27.8 %) and dry weight (33.3 %) when compared with culture medium without AC. However, many protocols used AC. For example, Faria et al. (2004a) used ½ MS supplemented with 1 g/l AC and 7 g/l agar for germination of 20 crosses and self-pollinated seed. The same culture medium was used by Lone et al. (2008) for the germination of seeds resulting from several D. phalaenopsis crosses.

Abiotic factors

The quality, quantity and periodicity of light are another set of factors that can significantly influence seed germination (Lin et al. 2011; Zeng et al. 2012; Parthibhan et al. 2012). In most studies, Dendrobium seed germination was possible at a temperature ranging from 22 to 28 °C (Fig. 6; Faria et al. 2004a; Lone et al. 2008; de Moraes et al. 2010; Galdiano-Júnior et al. 2011; Su et al. 2012; Soares et al. 2013) with a 9–16-h photoperiod under cool white fluorescent tubes (30.77 % of papers) in which the photosynthetic photon flux density (PPFD) was 10–60 µmol m−2 s−1 (Fig. 6; Xu and Yu 1984; Kong et al. 2007; Kananont et al. 2010; Table 1). Sometimes seed germinated in the dark (Meng et al. 2012), or at different intensities and quality of illumination [fluorescent white light; red light-emitting diodes (LEDs); blue LEDs; half red plus half blue (R:B = 1:1) LEDs; 67 % red plus 33 % blue (R:B = 2:1) LEDs; and 33 % red plus 67 % blue (R:B = 1:2) LEDs] at different developmental stages (Lin et al. 2011), or high PPFD (60 µmol m−2 s−1 by Kaewduangta and Reamkatog 2011 and Hossain et al. 2013b, or 150 µmol m−2 s−1 by Dutta et al. 2011). Ali et al. (2011) observed that continuous light resulted in highest seed germination of D. tetrachromum Rchb.f. (100 vs 48.9 % for a 16-h photoperiod) and D. hamaticalcar (97.5 vs 29.3 % for a 16-h photoperiod). de Moraes et al. (2010) used a 12-h photoperiod with a PPFD of 40 µmol m−2 s−1 for 180 days for seed germination of D. nobile. In fact, 43.4 % of papers used a 12-h photoperiod for the germination stage of Dendrobium species (Table 4). Early stages of germination were uniform from 0- to 24-h photoperiod in D. aqueum, but final germination and seedling production differed depending on photoperiod: increasing photoperiod from 8 to 24 h improved germination and seedling development from 48.5 to 96.0 % on ½ MS medium, but seeds germinated under continuous darkness showed lower germination (31.4 %), and seedlings failed to grow (Parthibhan et al. 2012). Soares et al. (2013) also used a 12-h photoperiod to germinate D. nobile seeds, but a low PPFD (13.5 µmol m−2 s−1). Galdiano-Júnior et al. (2011) used 75 µmol m−2 s−1 and a 16-h photoperiod for 90 days, obtaining D. nobile plants approximately 0.5 cm long and with two leaves after the subculture of seedlings germinated from seeds. Su et al. (2012) used a 16-h photoperiod with a PPFD of 17.55 µmol m−2 s−1 for 180 days during in vitro cultivation of Dendrobium (species not defined). Faria et al. (2004a) also used a 16-h photoperiod for D. nobile but PPFD was undefined, while Lone et al. (2008) used a PPFD of 27 µmol m−2 s−1 for D. phalaenopsis. Zhao et al. (2013a) observed that 92 % of D. wangliangii seeds germinated under a 16-h photoperiod and a PPFD of 36 µmol m−2 s−1 but seeds cultured under continuous darkness died, even after transfer from dark to light. Initial incubation of D. huoshanense seeds in the dark for 22 days, followed by transfer to low light intensity (1700 lux = 23 µmol m−2 s−1), resulted in 91 % of germinated seeds (Yang and Wang 1989).

Fig. 6
figure 6

Temperatures or range of temperatures used in papers on the asymbiotic germination of Dendrobium species, reported as percentages. Some temperature classes were joined as they lie within a defined range. NR not reported

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Table 4 Light intensity and photoperiod used in papers related to in vitro asymbiotic seed germination of Dendrobium species
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Other factors

Culture vessels

Orchid seeds germinate better in flasks than in culture tubes due to a greater volume of air (6- to 25-fold more) and thereby CO2 diffusion is greater when compared to culture tubes (Knudson 1922). Research on the influence of culture vessels or flask size with respect to orchid seed germination in vitro is still scant (Buffa Filho et al. 2002). de Moraes et al. (2010) tested the effect of flask size (100, 200 and 400 ml) on seedling growth. They observed that greatest height (2.24 cm), number of roots (2.41) and leaves (1.95), fresh (0.36 g) and dry (0.11 g) weight were obtained using 100-ml flasks, but seed germination per se was not tested, or quantified. Galdiano-Júnior et al. (2011) used 220-ml plastic flasks with 30 ml of culture medium. Some authors used 250-ml borosilicate flasks with 50 ml of culture medium for D. nobile (Faria et al. 2004a, b; Su et al. 2012) and D. phalaenopsis (Lone et al. 2008). Soares et al. (2013) used 600-ml flasks containing 80 ml of culture medium. These culture vessels were used for both seed germination and seedling growth.

Explant age

Normally, after pollination, about 100–140 days are required for Dendrobium seed to mature (Nimoto and Sagawa 1961). However, green pod culture or immature seed germination involves the use of green, undehisced capsules, which are harvested 70 days after pollination, opened and seeds are inoculated on culture media (Soundararajan 2009; Table 1). The time required for seed germination ranges from 30 to 59 days (Alam et al. 2002; Sunitibala and Kishor 2009; Parthibhan et al. 2012). Green pod seeds grown on Robert Ernst medium (Ernst 1982), KP medium (Knop 1865), KC medium, Curtis medium (Curtis 1936) and Pfeffer (Harvais 1972) medium became pale green or whitish green and eventually produced weak seedlings but on VW medium, KC medium, Thomale GD medium (Thomale 1954), Mitra et al. medium (Mitra et al. 1976) and Wolter and Skoog medium (WS; Wolter 1968) the seeds became brown and could not germinate. However, the addition of MS vitamins in WS medium resulted in comparatively better germination and seedling growth (e.g., Thomale GD and RE medium resulted in 67.7 and 48.4 % germination of D. aqueum seeds, whereas higher germination (71.9 and 58.0 %) and seedling growth was observed on the same medium when supplemented with MS vitamins) (Parthibhan et al. 2012; unpublished). Sharma et al. (2005) used VW medium to germinate 80–90 % of immature D. fimbriatum seeds from 70-day-old green, undehisced fruits which developed protocorms and later shoots, but only when 15 % CW and 0.1 mg/l NAA were added to the medium. Vijayakumar et al. (2012) also used immature seed germination via green pod culture for D. aggregatum, and observed that MS culture medium with 3 % sucrose, 1.5 mg/l BA and 15 % CW resulted in best germination, protocorm and shoot production (75 shoots/flask). Kumar et al. (2006) used KC green pod culture 70 days after pollination of D. chrysanthum and obtained 80–90 % seed germination, but that study was invalidated following a retraction.

Conclusions and future perspectives

Experiments and reviews on various factors that influence seed germination and seedling development in orchids, especially in Dendrobium, have been reported (Arditti 1967; Lo et al. 2004a, b; Saiprasad et al. 2004; Kauth et al. 2008; Mweetwa et al. 2008; Chugh et al. 2009; Ferreira et al. 2011). Moreover, nutritional and environmental conditions are species-specific. The environmental factors such as light exposure (photoperiod, light vs dark) and temperature will also influence seed germination both in nature and in vitro (Rasmussen 1995).

Asymbiotic germination of Dendrobium species is possible because of the large quantity of seeds produced per pod and the high rate of seed germinated in vitro. Nevertheless, abiotic conditions that are required for symbiotic Dendrobium seed germination are not unified and are often species-specific (Zhao et al. 2013b) and the use of established in vitro conditions provides a better environment for controlled asymbiotic seed growth and development. This would also allow for the identification of diverse factors and compounds that define successful germination and subsequent propagation of seedlings. Moreover, germinating seeds together with a species-specific mycorrhizal fungus could improve the success of seed-based conservation programs (Teixeira da Silva et al. 2015b), both in in situ germplasm conservation and in reintroduction efforts (Keel et al. 2011).

A meta-analysis of the Dendrobium asymbiotic seed germination literature reveals that 37 Dendrobium genotypes (including species and hybrids) were used in studies of asymbiotic germination, the most frequent being D. candidum (D. officinale) (Table 2). Mature seeds are usually used to start Dendrobium in vitro germination (Table 2). EtOH and HgCl2, EtOH and NaOCl, and EtOH and flaming were the most frequently used procedures for seeds and fruit sterilization (Table 3). MS and ½ MS were the most common basal culture medium (Fig. 3). Most papers used no PGRs or additives in basal media (Figs. 4, 5). As abiotic factors, 25 ± 2 °C as temperature (Fig. 6), a 12-h photoperiod, 1000–2200 lux, and 30–42 µmol m−2 s−1 light intensity (white fluorescent lamps) were reported as the most favorable conditions (Table 4). The application of optimized conditions leads to the successful development of seeds through an established set of phases (Fig. 7).

Fig. 7
figure 7

Asymbiotic seed germination stages in Dendrobium aqueum (Lindley) cultured on half-strength (macronutrients) MS medium (devoid of PGRs) under 16-h photoperiod at 23 ± 2 °C (more details in Parthibhan et al. 2012). a Imbibed, enlarged seeds (zygotic embryos) with testa, turned into green (photosynthetic) ×20 after 15 days of culture. b Enlarged seeds (zygotic embryos) with half-ruptured testa after 35 days of culture. c Enlarged seeds (zygotic embryos) without a testa after 57 days after culture. d Protocorms with green pointed shoots 75 days after culture. e Protocorms with developed shoots and rhizoids after 107 days after culture. f Seedlings with shoots and developed roots after 126 days after culture. Unpublished photos (Parthibhan et al.)

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This review may serve as a detailed handbook, summarizing the studies on Dendrobium asymbiotic germination; thus being useful for both experienced researches and Dendrobium amateurs, since there are no ideal parameters for germination of diverse species and hybrids of the genus Dendrobium.

Author contribution statement

All authors contributed equally to all aspects related to the manuscript.