Introduction

Orchidaceae consists of 30,000–35,000 species in 850 genera and is one of the largest families of flowering plants (Hossain et al. 2013). In India, with a wide range of climatic conditions, there are at least 1331 species of orchids representing 186 genera (Misra 2007). Several important orchid species are listed as endangered and under threat of extinction in the Red Data Book of the International Union for Conservation of Nature (IUCN; Nayar and Sastry 2000). Climate change, deforestation, grazing, over-exploitation for medicinal and horticultural uses, and lack of suitable pollinators (Swarts and Dixon 2009; Barman and Devadas 2013; Pant 2013) are major factors affecting survival and propagation of orchids in the wild.

The genus Anoectochilus is popularly known as “Jewel Orchids” and “King of Medicine” in China and Taiwan because of the beautiful foliage and medicinal properties of plants in this genus (Gutierrez 2010). Anoectochilus species produce a wide range of biological compounds, including an alkaloid called kinsenoside that is used to treat diabetes, hyperliposis, and breast cancer (Du et al. 2001; Shyur et al. 2004; Zhang et al. 2007). An aqueous extract of Anoectochilus formosanus stimulated immunity (Lin and Hsieh 2005) and showed hepatoprotective activity (Wu et al. 2007). In vitro studies are limited in this valuable genus, with reports of successful propagation for only a few species and limited to shoot tip and nodal explants (Gangaprasad et al. 2000; Ket et al. 2004; Sherif et al. 2012; Zhang et al. 2015).

Anoectochilus elatus is an endangered monopodial terrestrial jewel orchid native to India and distributed throughout the Eastern and Western Ghats of Tamil Nadu, and Kerala. It has various vernacular names including Mayilraegai saedi and Kairaegai saedi in Tamil, and Nagathali in Malayalam. Tribal people use this plant to cure chest and abdominal pains and to treat snake bites (Sarkar 2012). In recent years, rapid deforestation, soil erosion, high temperatures, and scarcity of pollinators have created problems for this species. The wild population is under depletion due to these disturbances in the natural regeneration process (Sherif et al. 2012).

There are no reports on indirect organogenesis in this genus; therefore, the objective of this study was to develop an indirect de novo organogenesis of A. elatus from various explant sources.

Materials and methods

Explant source

Three different juvenile explants (node [1 cm], internode [1.5 cm], and leaf [1.5 cm2]) were excised from 6-mo-old in vitro-raised plants of A. elatus (Sherif et al. 2012).

Culture medium and conditions

For callus production and shoot regeneration, explants were cultured on Mitra medium (Mitra et al. 1976) supplemented with 20 g L−1 sucrose and different plant growth regulators (PGRs), including the cytokinins N6-benzyl adenine (BA), kinetin (KN), thidiazuron (TDZ), N6-(2-isopentyl) adenine (2ip), and zeatin (ZEA) and the auxins 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 4-amino-3,4,6-trichloro picolinic acid (Pic), at concentrations ranging from 0.1 to 3.0 mg L−1, either alone or in combination. In addition to the PGRs mentioned above, various antioxidants such as citric acid and trisodium citrate at 50–200 mg L−1 and complex elicitors such as peptone (50–200 mg L−1), coconut water (5–20 %), potato extract (5–20 %), and banana pulp (5–20 g L−1) were added to the medium for multiple shoot formation. Healthy elongated shoots (≥4 cm) separated from multiple shoot clumps were cultured on Mitra medium supplemented with silver nitrate (AgNO3) at different concentrations (0.1–2.0 mg L−1) for rooting. The pH of the medium was adjusted to 5.7 using 0.1 N NaOH or HCl before adding 2 g L−1 PhytagelTM as a solidifying agent. Prepared medium was dispersed into culture tubes (25 × 150 mm), 100-ml saline bottles (4.5 × 9 cm), or 250-ml conical flasks (Borosil®, Chennai, India) prior to autoclaving at 121°C for 15 min. All cultures were maintained under a 16-h photoperiod with cool white fluorescent tubes (Philips TL–D Super 80, Gurgaon, India; 40 μmol m−2 s−1) at a constant temperature of 23 ± 2°C. All chemicals were purchased from Hi-Media® Pvt. Ltd. (Mumbai, India).

Acclimatization

Rooted shoots were removed from the rooting medium and washed thoroughly in distilled water to remove any adhering medium. The plantlets were then transferred to eco-friendly paper cups (6-cm diameter; Sai Papers, Tiruchirappalli, India) containing an autoclaved mixture of garden soil, sand, vermicompost, and used tea waste in a ratio of 8:4:2:1 (w/w/w/w). The plantlets were covered with transparent polyethylene bags (12 × 8 cm in size, Tarson® Products, New Delhi, India) in order to reduce fungal load and other infections and to maintain humidity, and watered with half-strength Mitra solution without sucrose once every 4 d for 3 mo under controlled conditions (24 ± 2°C, 80–85% relative humidity, 16-h photoperiod, and 40 μmol m−2 s−1 light intensity). After the first 2 mo, the polyethylene bags were removed and the humidity was gradually reduced to 60–70 %. The plants were then transferred to a greenhouse and finally transported to the National Orchidarium (Yercaud), Tamil Nadu, for further establishment.

Data collection and statistical analysis

All experiments were conducted as a completely randomized design. Each experiment included seven replicates, and experiments were performed three times. Data on callus morphological features, callus induction percentage, and shoot multiplication (number of shoots per callus and mean shoot length per explant) were collected every week for 16 wk, and the number of roots and average root length per explant were recorded for 6 wk. The data were analyzed by analysis of variance (ANOVA), and significant differences between treatments were determined based on Duncan’s multiple range test at p < 0.05 using SPSS-PASW statistical program version 18.0.0 (SPSS, Chicago, IL).

Results

Callus induction and proliferation

Figure 1 shows the complete series of callus initiation and shoot development from nodal (Fig. 1a–f ), internodal (Fig. 1g–l ), and leaf explants (Fig. 1m–r ). The explants began to enlarge within 2 wk of culture, and callus developed from the cut ends after 4 wk. Callus induction occurred on all explants (node, internode, and leaf) after 8 wk of culture (Fig. 1a, g, n ). The callus was greenish-yellow, compact, and nodular in texture (Fig. 1c, h, p ). The efficacy of callus induction varied with explant type. Among the different cytokinins and auxins tested individually, the maximum callus initiation percentage was observed on Mitra medium containing 1.0 mg L−1 TDZ. The best callus initiation response was achieved from internode explants (68.3 %) followed by node (55.5 %) and leaf (53.8 %) explants (Table 1). To increase callus formation and proliferation, combinations of TDZ, BA, and NAA were tested to look for possible synergistic effects. After 16 wk of culture, Mitra medium with 1.0 mg L−1 TDZ and 0.5 mg L−1 NAA showed the best response, with callus initiation frequencies of 77.8, 69.7 and 64.2% for internode, node and leaf explants, respectively (Table 1).

Figure 1
figure 1

Indirect organogenesis of Anoectochilus elatus. (a–f) Callus initiation and shoot development from nodal explant. (g–l) Callus initiation and shoot development from internodal explant. (m–r) Callus initiation and shoot development from leaf explant. (s) In vitro rooting. (t) Hardening of regenerated plantlets covered with transparent polyethylene bags. (u) Hardened plants in greenhouse. (v) Well-acclimatized plants maintained in the National Orchidarium, Yercaud.

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Table 1 Callus induction and proliferation from node, internode and leaf explants of Anoectochilus elatus on Mitra medium supplemented with various plant growth regulators, alone and in combination, after 16 wk of culture
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Shoot bud initiation and multiplication

Shoot bud regeneration from callus of all three explant types was observed on Mitra medium fortified with different PGRs at different concentrations. Calluses with tiny shoot buds were cultured on Mitra medium supplemented with individual cytokinins (BA, KN, 2ip, and ZEA) and auxins (NAA, IBA, and IAA) at different concentrations (Fig. 1d, j, q ). After 12 wk of culture, the 1.0 mg L−1 BA and 0.5 mg L−1 NAA treatments showed the greatest number of shoots per explant from callus derived from all three explant types. The greatest numbers of shoots (11.3 per callus with 1.0 mg L−1 BA and 12.2 per callus with 0.5 mg L−1 NAA) were obtained using callus derived from internodal explants, followed by node-derived callus (9.5 shoots/callus on 1.0 mg L−1 BA and 8.3 shoots/callus on 0.5 mg L−1 NAA) and leaf-derived callus (8.1 shoots/callus on 1.0 mg L−1 BA or 2.0 mg L−1 BA or 0.5 mg L−1 NAA; Table 2).

Table 2 Shoot bud induction and multiplication from callus derived from node, internode and leaf explants of Anoectochilus elatus on Mitra medium supplemented with various plant growth regulators alone or in combination, after 12 wk of culture
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To increase the number of shoots per callus, different concentrations of NAA in combination with 1.0 mg L−1 BA were tested. After 12 wk of culture, there was a marked increase in shoot frequency when auxin was combined with cytokinin. The highest mean number of shoots per explant was observed from callus derived from internodal explants (23.8 shoots/callus), followed by node (19.4 shoots/callus) and leaf (18.2 shoots/callus), on Mitra medium supplemented with 1.0 mg L−1 BA and 0.5 mg L−1 NAA (Table 2).

The effect of antioxidants (citric acid and trisodium citrate) and organic supplements (peptone, coconut water, potato extract, and banana pulp) on shoot multiplication was tested using Mitra medium containing 1.0 mg L−1 BA and 0.5 mg L−1 NAA. Of the two antioxidants tested, 100 mg L−1 trisodium citrate supported the best shoot formation response. After 12 wk of culture, 30.6 shoots/callus were obtained from internode explants, followed by 28.9 from node and 25.5 from leaf explants (Table 3). Of the organic supplements tested, coconut water supported the greatest increase in shoot production, followed by peptone. Using 10% coconut water, a maximum 41.8 shoots/callus were obtained from internode explants, followed by 32.9 and 29.7 from node and leaf explants, respectively (Fig. 1f, l, r and Table 3).

Table 3 Multiple shoot proliferation from callus derived from node, internode and leaf explants of Anoectochilus elatus on Mitra medium supplemented with 1.0 mg L−1 BA + 0.5 mg L−1 NAA and additives, after 12 wk of culture
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In vitro rooting and acclimatization

In vitro-raised shoots (≥4 cm) were transferred to Mitra medium supplemented with different concentrations of AgNO3. The effect of AgNO3 on in vitro rooting response varied with concentration. The optimum concentration of AgNO3 was 1.0 mg L−1, on which 97.8% of shoots produced roots, with an average of 3.2 roots/shoot and an average root length of 2.1 cm, after 6 wk (Fig. 1s and Table 4). Rooted shoots with two to three leaves were acclimatized in paper cups covered with transparent polyethylene bags and maintained at 24 ± 2°C and 80–85% humidity (Fig. 1t ). After 2 mo, the bags were removed and the humidity was gradually reduced to 60–70 %. Plantlets were successfully established in the greenhouse with a 72.3% survival rate (Fig. 1u ). After 2 mo, under greenhouse conditions, the plants were transferred to the National Orchidarium, Yercaud, Tamil Nadu, for further establishment in the field (Fig. 1v ).

Table 4 Root induction of shoots from callus of node, internode and leaf explants of Anoectochilus elatus on Mitra medium supplemented with AgNO3, after 6 wk of culture
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Discussion

In general, regeneration in orchids is accomplished using asymbiotic or symbiotic seeds germinated in vitro (Kumaria and Tandon 1991). In vitro regeneration of wild orchids using different explants has proven challenging regardless type of the explant used. In this present system, efficient plant regeneration via indirect organogenesis has been developed from calluses derived from node, internode, and leaf. Since callus is a potential source for regeneration, by virtue of having the capacity to form many meristematic regions (George 1996), it can play an important role in genetic transformation studies and secondary metabolite production, and also used to produce abiotic stress tolerant lines (Sarangi et al. 2011; Bakrudeen et al. 2012).

Callus induction and proliferation

The in vitro-derived explants possess early stages of morphogenesis compared to tissue excised from in vivo explants (George 1996). The induction of callus was mainly influenced by PGRs. Among the individual PGRs tested, TDZ induced maximum callus formation from all explant types, followed by BA and NAA. The internodal explants showed the maximum callus formation and proliferation, followed by node and leaf explants. TDZ is involved either directly or indirectly in several morphological and physiological responses in plant tissues (Guo et al. 2011). TDZ and NAA are reported to promote the formation of protocorm-like bodies, callus, and shoots from different explants, and from different species of terrestrial and epiphytic orchids including Phalaenopsis and Doritaenopsis (Ernst 1994), Cymbidium sinense (Chang and Chang 2000), Doritaenopsis (Park et al. 2003), Vanilla planifolia (Giridhar and Ravishankar 2004), Phalaenopsis gigantea (Latip et al. 2010), Oncidium flexuosum (Mayer et al. 2010), and Xenikophyton smeeanum (Mulgund et al. 2011). As a whole, these studies reveal that cytokinin combined with a low level of auxin is important for callus initiation and proliferation using various explants.

Shoot bud initiation and multiplication

Not surprisingly, the ability of callus to regenerate varied with the concentration and combination of auxins and cytokinins. In this study, the optimal individual concentration of cytokinin combined with a lower level of auxin stimulated shoot initiation. The presence of BA and NAA in the medium suppressed proliferation of callus and played an important role in shoot bud induction. Although BA stimulated shoot buds of A. elatus when present alone, BA in combination with an auxin (NAA) further enhanced shoot multiplication, as previously reported in several other orchids (Vij and Pathak 1990; Murthy and Pyati 2001; Sheelavanthmath et al. 2005).

Antioxidant addition to the regeneration medium increased shoot number over that obtained from regeneration medium amended with PGRs alone. Trisodium citrate is a salt of CA that promotes shoot multiplication in combination with BA and NAA. In regeneration medium containing either trisodium citrate or citric acid, callus necrosis and browning were substantially reduced and healthy shoots were formed.

Apart from the influence of PGRs, coconut water played a significant role due to its growth-promoting property (Molnar et al. 2011; Kaur and Bhutani 2012) when compared to other supplements such as potato extract and banana pulp (Shadang et al. 2007; Gnasekaran et al. 2012).

In vitro rooting and acclimatization

The results reported here indicate that AgNO3 can be used to induce roots. Silver nitrate is involved in several pathways pertaining to polyamines, ethylene, and calcium-mediated metabolism in plant cells (Pua and Chi 1993; Kumar et al. 2009). AgNO3 blocks the action of ethylene in plants and is widely used in plant tissue culture to promote regeneration, multiplication, and root formation in dicotyledonous species such as Vitex negundo and Decalepis hamiltonii and in orchid species such as Vanilla planifolia (Ganesh et al. 1996; Khalafalla and Hattori 2000; Giridhar et al. 2001; Kumar et al. 2009; Steephen et al. 2010).

Plantlets grown in vitro have been continuously exposed to a unique environment to provide minimal stress and optimum conditions for plant multiplication. Plantlets develop within the culture vessels under a low level of light, under aseptic conditions, and on a medium containing sugar and nutrients to allow for heterotrophic growth compared to ex vitro conditions (Pospisilova et al. 1999; Sherif et al. 2012). Because of these conditions, in vitro-raised plants may quickly wilt during transplantation to greenhouse or field conditions (Hiren et al. 2004; Lavanya et al. 2009). Another reason for wilting is the absence of a waxy cuticle on plants raised in vitro compared to ex vitro plants (Gilly et al. 1997). Thus, by using the polythene bag method, 72.3% of the plantlets were successfully acclimatized to greenhouse conditions. This method was successful and agreed with previous results from this same species (Sherif et al. 2012) and similar results from A. formosanus (Ket et al. 2004). The well-stabilized plants were handed over to the National Orchidarium, Yercaud, for further maintenance and conservation of this valuable endangered orchid.

Conclusions

This is the first report describing a protocol for callus morphogenesis and de novo organogenesis from three different explants of A. elatus. Among the three explants tested, internodal explants responded the best. The addition of an antioxidant (100 mg L−1 trisodium citrate) and an organic supplement (10% coconut water) to the regeneration medium increased multiple shoot formation and elongation. Effective rooting was achieved with the addition of 1.0 mg L−1 AgNO3. This protocol should be useful for large-scale multiplication and conservation of this species and for other research and commercial applications.