Acta Physiol Plant (2013) 35:829–840 DOI 10.1007/s11738-012-1127-3 ORIGINAL PAPER Asymbiotic seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal fungus on seedling development G. Mahendran • V. Muniappan • M. Ashwini T. Muthukumar • V. Narmatha Bai • Received: 21 February 2012 / Revised: 11 October 2012 / Accepted: 15 October 2012 / Published online: 4 November 2012 Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2012 Abstract An in vitro plant regeneration protocol was successfully established for Cymbidium bicolor an epiphytic orchid by culturing seeds from green pods. Immature seeds were germinated on four basal media viz., Murashige and Skoog (MS) medium, Knudson C (KC) orchid medium, Knudson C modified Morel (KCM) medium and Lindemann orchid (LO) medium. Seed germination and protocorm development was significantly higher in LO medium (96.6 %) followed by KCM (89 %), MS (77.5 %) and KC (62.7 %) media after 56 days. For multiple shoot induction the protocorms were transferred to B5 medium supplemented with cytokinin. Among the various cytokinins tested, BAP (4.42 lM) induced maximum (27.59) number of multiple shoots per explant. IBA was effective in inducing healthy roots. Tissue-cultured protocorms and seedlings of C. bicolor were inoculated with AC-01 fungal strain (Moniliopsis sp.) isolated from the mycorrizal roots of an epiphytic orchid Aerides crispum. Mycorrhizal fungi significantly enhanced number of roots, root length and shoot number. Keywords Cymbidium bicolor
Asymbiotic seed germination
Protocorm
Multiple shoots
Aerides crispum
Mycorrhiza
Fungal co-culture Communicated by J. van Staden. G. Mahendran (&) Department of Botany, Plant Tissue culture lab, Bharathiar University, Coimbatore 641046, India e-mail: mahendran0007@gmail.com V. Muniappan
M. Ashwini
T. Muthukumar
V. Narmatha Bai Department of Botany, School of Life Sciences, Bharathiar University, Coimbatore 641046, Tamilnadu, India Abbreviations BAP Benzyl amino purine KIN Kinetin TDZ Thidazuron –N phenyl-N0 -1,2,3-thadiazol-5-ylurea LO Lindemann orchid medium KCM Knunson C modified Morel medium NAA a-Napthalene acetic acid MS Murashige and Skoog medium KC Knudson C orchid medium a.s.l Above sea level Introduction Orchidaceae is one of the largest and advanced plant families with 20,000–30,000 species (Chugh et al. 2009). Orchids are fascinating groups of ornamental plants with numerous exotic novel cultivars possessing elegant flowers produced by interspecific, as well as intergeneric hybridization. However, orchids in the wild are endangered as a consequence of environmental disruption, succession of natural habitats and over exploitation for horticultural purposes. In situ conservation of dwindling populations of endangered orchid species is very difficult because of their slow growth and low seed germination. Thus, in recent years, the maintenance of living collections of orchids has been considered to be an important aspect of conservation (Sharrock 2007; Somiya 2007). All orchids have an obligate relationship with mycorrhizal fungi during seed germination and development (Rasmussen 1995). The mycorrhizal fungi provide the carbon source necessary for seed germination and seedling establishment (Yam and Arditti 2009). In some green and achlorophyllous orchids, the dependency on mycorrhizal 123 830 fungi may extend into adulthood (Julou et al. 2005; Abadie et al. 2006; Rasmussen and Rasmussen 2007). Moreover, the application of mycorrhizal fungi in horticulture and for conservation purposes has recently gained considerable attention (Rasmussen 2002; Rasmussen and Rasmussen 2007; Swarts and Dixon 2009; Zettler et al. 2007). Cymbidium or ‘‘boat orchid’’ is a popular orchid grown commercially worldwide (Chugh et al. 2009). Today, orchids such as Cymbidium, Dendrobium, Oncidium and Phalaenopsis are marketed globally and the orchid industry contributes substantially to the economy of many the South East Asian countries. Cymbidium bicolor Lindl. is an important horticultural orchid known for its beautiful flowers (Chugh et al. 2009). In Cymbidium, plantlets were regenerated in in vitro using shoot tips (Morel 1964), mature and immature seeds (Chung et al. 1985; Shimasaki and Uemoto 1990), green capsules (Hossain et al. 2010; Deb and Pongener 2011), flower stalks (Wang 1988), pseudo bulbs (Shimasaki and Uemoto 1990), shoot segments (Nayak et al. 1997), flower buds (Shimasaki and Uemoto 1990), protocorm-like bodies (PLBs) (Begum et al. 1994a; Huan and Tanaka 2004; Teixeira da Silva et al. 2007), thin cell layers of PLBs (Malabadi et al. 2008), artificial seeds (Nhut et al. 2005) and through somatic embryogenesis (Begum et al. 1994b; Chang and Chang 1998; Huan and Tanaka 2004; Mahendran and Narmatha Bai 2012). Hoque et al. (1994) found that Phytomax medium was the best among the five different media (Phytomax, Modified Vacin and Went, KC, KCM and LO medium) tested for large scale multiplication of C. bicolor. Transplantation stage continues to be a major bottleneck in the micropropagation of orchids. A substantial number of micropropagated plantlets fail to survive when transferred from in vitro conditions to a greenhouse or field environment. The greenhouse or field environment have substantially lower relative humidity, higher and intense light levels that are stressful to micropropagated plants compared to in vitro conditions (Chugh et al. 2009). The manipulation of acclimatization conditions reduce the plantlet losses, however, it incurs an additional production cost. Attempts have been made to reduce transplantation losses by the use of CO2 enriched environment and high light intensity for autotrophic miropropagation (Vyas and Purohit 2003; Dave and Purohit 2004). This technology is yet to gain popularity (Nowak 1998). Alternatively the naturally occurring beneficial mycorrhizal association between fungi and orchids could be exploited for better nutrient acquisition (Zhu and Miller 2003), improved plant water relations and imparting tolerance to abiotic stresses such as drought and salinity (Kapoor et al. 2008) in in vitro derived plantlets. Such biologically hardened plants could perform better and consequently, reduce losses due to environmental stresses. 123 Acta Physiol Plant (2013) 35:829–840 Previous studies have shown that mycorrhization of tissue-cultured orchid plantlets reported to enhance, plant height, stem diameter, root number, biomass, plant survival and establishment percentages (Chang and Chou 2007; Fang et al. 2008; Hou and Guo 2009). Mass propagation for commercial cultivation requires a simple, economical, rapidly multiplying and highly reproducible protocol. Keeping these in mind the present study was carried out to (a) select the appropriate medium for seed germination and protocorm development (b) induce multiple shoot buds from seed derived protocorm and (c) test the effectiveness of the mycorrhizal fungus on the growth and development of protocorm and seedling development under in vitro condition and consequently on the ex vitro acclimatization process. Materials and method Preparation of green capsules Green capsules of C. bicolor (naturally pollinated) were collected from National Orchidarium, Yercaud (11°480 N latitude 10°550 and 78°-130 E longitude, 1,500 m a.s.l), Salem, Tamilnadu, India. The freshly collected capsules were surface sterilized in 0.01 % (w/v) mercuric chloride solution for 2 min, rinsed thoroughly thrice with sterile distilled water, dipped in 70 % (v/v) ethanol for 30 s and flamed. Seeds from the surface sterilized capsules were extracted by longitudinally splitting the capsule with a sharp sterilized surgical blade. The seeds were then spread as thin film in the test tube containing 20 ml of culture media. The cultures were maintained at 25 ± 2 °C under cool white fluorescent tubes at a light intensity of 50 lmol m-2 s-1 with 16/8 h L/D photoperiod in culture room conditions. Seed viability test Seed viability was tested according to Vellupillai et al. (1997). For enumerating the seed viability percentage, seeds from the fresh capsules were treated with 1 % (w/v) 2,3,5-triphenyl tetrazolium chloride (pH 7.0) in the dark overnight. Treated seeds were observed with a light microscope and scored as either viable (red embryo) or nonviable (white embryo). Asymbiotic seed culture Four asymbiotic orchid seed germination media namely MS (Murashige and Skoog 1962), KC (Knudson 1946), KCM (Morel 1965) and Lindemann orchid (LO) (Lindemann et al. 1970) were tested to select a suitable medium Acta Physiol Plant (2013) 35:829–840 for seed germination. All the media were supplemented with 3 % (w/v) sucrose and solidified with 0.8 % (w/v) agar (Hi media-India).The pH of the media was adjusted to 5.6–5.8 with 1 N NaOH or HCl before autoclaving at 121 °C, 105 kPa for 20 min. Induction of multiple shoots Lindemann orchid medium was supplemented with cytokinins such as BAP, KIN and TDZ for multiple shoot induction. The media turned brown and arrested the seedling development. So, the protocorms (60-days old) obtained from LO medium were subcultured in B5 medium supplemented with cytokinins including BAP (1.10, 2.21, 4.42, 8.84 or 13.26 lM), Kin (1.16, 2.32, 4.64, 9.28 or 13.92 lM) and TDZ (1.13, 2.26, 4.52, 9.24 or 13.76 lM) individually to asses effect on multiple shoot development. Rooting For root development, individual shoots each with 2–3 expanded leaves were detached from the shoot clumps and transferred to B5 liquid medium containing either IBA (4.92–14.76 lM) or NAA (5.37–16.11 lM). Fungal isolation and morphological characterization Fresh roots of the epiphytic orchid Aerides crispum Lindl. were collected from Vellingiri hill (longitude 6°400 and 7°100 E and latitude 10°550 and 11°100 N, 1,400 m a.s.l) Coimbatore, Tamilnadu, India. Mycorrhizal fungus (designated as AC-01) was isolated using modification of Masuhara and Katsuya (1994) method. The roots were soaked in teepol detergent solution for about 5–6 min and thoroughly washed with running water. The roots were surface sterilized with 0.05 % (w/v) mercuric chloride for 2 min and washed 3–7 times with sterile distilled water. The roots were then cut into longitudinal thin sections after the removal of the velamen tissue and transferred on to potato dextrose agar (PDA) medium containing 100 mg/l streptomycin. After 10 days of incubation at 30 °C, the hyphal tips emerging from the root segments were transferred onto fresh PDA medium. The fungal isolate was maintained on PDA at 30 °C in the dark. The diameter of five colonies were measured daily for 2 weeks. Growth rate was calculated by plotting colony diameters against time (Nontachiyapoom et al. 2010). For microscopic observation, the fungal hyphae were stained in trypan blue (0.005 %) (w/v) lacto glycerol solution and mounted in clean lacto glycerol and examined under an Olympus BX51 compound microscope attached with a ProgRes C3 camera. The number of nuclei per fungal cell was enumerated as per Leonerd (1979). The fungal 831 taxon was identified using morphological and growth characteristics as described by Currah et al. (1997). Orchid mycorrhizal fungus inoculation The protocorms (60-days old) and seedlings (120-days old) of C. bicolor were implanted on B5 Basal medium containing 2 % sucrose and 0.8 % agar for co-cultivation in test tube (protocorm) and conical flasks (seedling). One PDA agar (6 mm in diameter) disc containing the fungal mycelium was taken from the margin of the fungal colony and placed 2 cm away from the protocorm or seedling. Uninoculated protocorms (60-days old) or seedlings (120days old) served as control. All treatments were maintained under fluorescent lights (50 lmol m-2 s-1) with a 16-h photoperiod at 25 ± 2 °C. The influence of the orchid mycorrhizal fungus on plant growth parameters such as plant height, number of new roots, root length, number of new shoots per protocorm or seedlings were assessed 125 days after inoculation. Each treatment had ten replicates and the experiment repeated thrice. Estimation of fungal colonization Free-hand transverse sections of the roots were made and stained with trypan blue. Stained sections were mounted and observed for presence of mycorrhizal structures (pelotons). To determine the ratio of cortical cells containing pelotons, the total number of cortical cells per microscopic field (five microscopic fields were examined for each section) and the number of cortical cells containing intact and lysed pelotons were counted and recorded. Pelotons with distinguishable hyphae were considered intact and those in which the hyphae were indistinguishable were considered lysed. Ex vitro plant establishment For ex vitro establishment, well-rooted mycorrhizal and non-mycorrhizal plantlets were rinsed thoroughly with tap water to remove residual nutrients from the plant body and transplanted into plastic pot (10 9 8 cm; height 9 diameter) containing sterilized vermiculite (50 g per pot). The plastic pots were covered by polyethylene bags and maintained under fluorescent lights at 50 lmol-l m-2 s-1 with a 16-h photoperiod at 23 ± 2 °C. Experimental design and data analysis The germination percentage was recorded at 14, 28, 42 and 56 days of culture. Percent germination was calculated by dividing the number of germinated seeds by the total number of seeds observed. Number of shoots, height, root 123 832 Acta Physiol Plant (2013) 35:829–840 number and root length was recorded after 56 days of culture. Each treatment was repeated thrice with ten replicates. Data were subjected to analysis of variance (ANOVA) and the mean values were separate using Duncan’s Multiple Range Test (DMRT). One and two-sample t test was used to compare the effects of the presence of the orchid mycorrhizal fungus in protocorms and seedlings. The statistical package SPSS (Version-17) was used for the analyses. Results Tetrazolium (TZ) viability test indicated a mean embryo viability of 73 %. The embryos enlarged and occupied the entire seed coat after 14 days of culture (Fig. 2a). Germination as evidenced by enlargement of the embryo was first observed in LO medium, followed by both KCM and MS medium. The seed germination was delayed in KC medium by 42 days. After 42 days, the embryos by repeated cell divisions emerged rupturing the testa in KCM medium (Fig. 2b). The percentage seed germination was 73.5 % in KCM basal medium, 55.6 % in MS medium and 43.5 % in KC medium. In LM the percentage seed germination was 89.2 % (Fig. 1). The embryos in LO medium swelled and transformed into globular hairy protocorm (Fig. 2c), which subsequently produced promeristem. The germination process was initially erratic and inconsistent, but the percentage of germination increased gradually and reached the significantly highest value at 56 days after culture. Seed germination was 96.6 % in LO medium followed by 89 % in KCM medium, 77.5 % in MS medium, and 62.7 % in KC medium. After 56 days, a pair of leaves emerged from the surface of the protocorms. MS and KCM basal media supported protocorm development after 56 days of 120 Seed germination (%) KC KCM MS LO Fig. 2 Asymbiotic seed germination, multiple shoot development c and effect of mycorrhiza (AC-01) on seedling development in Cymbidium bicolor. a Formation of protocorms on Lindmann medium after 14 days inoculation (bar 150 lm). b Embryo emerging out of the seed by rupturing the testa on Lindemann orchid medium (bar 100 lm). c 56 days old protocorms developed on LM orchid medium (bar 0.5 cm). d Formation of multiple shoots on B5 supplemented with 13.92 lM of KIN (bar 0.5 cm). e Multiple shoots developed on B5 medium supplemented with 4.42 lM of BAP (bar 0.5 cm). f Rooting of micropropagated plant on B5 medium supplemented with 14.76 lM of IBA (bar 0.5 cm). g Effect of isolated orchid mycorrhiza (AC-01) on protocorm (bar 0.5 cm). h Influence of fungus on new shoot formation after 4 months of mycorrhizal inoculation (bar 1.0 cm). i Influence of fungus on root growth (bar 1.0 cm). j Hardened plantlet after 2 months under ex vitro condition (bar 2.0 cm). E embryo. S seed coat inoculation, whereas protocorm development was observed only after 70 days of culture in KC medium. When LO medium was supplemented with cytokinins such as BAP, KIN and TDZ for multiple shoot induction, the media turned brown with time possibly indicating the phenolic exudation. Browning of the media coincided with the arrest of seedling development, so the seedlings were transferred to B5 medium supplemented with three different cytokinins such as BA or TDZ or Kin individually (Table 1). Two shoots were formed in B5 medium devoid of growth regulators at 90 days. Among the three cytokinins tested, multiple shoot induction was frequent in BAP (4.42 lM), followed by Kin (13.92 lM). Among the different levels BAP tested, the maximum number of shoots was observed on the B5 medium containing 4.42 lM of BAP (27.59 ± 0.88) (Table 1; Fig. 2d, e). The shoot buds first appeared as small white protuberances over the surface of the protocorm which eventually developed into multiple shoots within 35–56 days. The number of shoot buds increased with increasing concentration of BAP up to an optimal level of 4.42 lM. Among the various concentrations of Kin and TDZ tested, maximum number of the multiple shoots were recorded in B5 medium supplemented with 13.92 lM Kin (20.75 ± 0.35 shoots/explant) and 13.76 lM TDZ (5.25 ± 0.31 shoots/explant). 100 Rooting 80 IBA significantly promoted all the rooting parameters, including root length and number of root (Table 2; Fig. 2f). Significantly higher root numbers were recorded in 14.76 lM of IBA (5.40 ± 0.24). 60 40 20 Characterization of the fungal morphology 0 14 28 42 56 Number of days Fig. 1 Effects of different culture media on seed germination of Cymbidium bicolor. Error bars indicates ±SE 123 Colonies of the isolate AC01 cultured on PDA and identified as Moniliopsis sp. grew at the rate of 3.41 mm per day. The colonies were effuse, dirty white and turning brown with age. The mycelium was partly superficial and Acta Physiol Plant (2013) 35:829–840 833 123 834 Acta Physiol Plant (2013) 35:829–840 Table 1 Effect of various cytokinins on multiple shoot induction from seed derived protocorms of Cymbidium bicolor BA (lM/l) Kin (lM/l) TDZ (lM/l) No of multiple shoots Shoot length (cm) – – – 2.20 ± 0.20i 3.58 ± 0.19d,e – e 1.10 – 2.21 – 4.42 – 19.80 ± 0.37 2.98 ± 0.83f,g 13.26 – – 18.60 ± 0.50c 3.74 ± 0.78c,d – 3.40 ± 0.24 g,h 4.14 ± 0.11b,c 5.20 ± 0.20 f 4.76 ± 0.12a 1.16 2.32 4.64 3.34 ± 0.27d,e,f d 10.40 ± 0.50 – 15.60 ± 0.40 3.20 ± 0.27e,f – 13.92 – 20.40 ± 0.24b 2.02 ± 0.14j – – – – – – – – – – F-value15, 64 No of roots/plant Root length/ plant (cm) NAA (lM/l) 4.92 – 3.40 ± 0.24c,d 3.48 ± 0.34c 9.84 – b,c 4.00 ± 0.00 4.70 ± 0.15b 14.76 – 5.37 5.40 ± 0.24a 2.40 ± 0.24e 5.82 ± 0.14a 2.84 ± 0.15d 10.74 3.20 ± 0.20d 3.78 ± 0.21c 16.11 b 4.20 ± 0.20 4.48 ± 0.37b 24.15*** 88.87*** *** Significant at P \ 0.001 Values represent mean ± SE of three repeated experiments each with 10 replications. Means in a column with the different letter (superscript) are significantly different according to DMRT (P \ 0.05) partly immersed in the medium (Fig. 3a, b). The hyphae were regularly septate, brown, 4.37–(5.52)–8.05 lm wide and multinucleate (5–8 per cell). Rhizomorphs and clamp connections were absent. Monilioid cells when present were terminal short chains and ellipsoidal [6.9–(12.96)– 23.0 9 6.9–(11.08)–16.1 lm] (Fig. 3c, d). Numerous cluster of sclerotia formed a brown crust on the colony surface (Fig. 3e, f). Fungal effects on plant growth At the time of harvest (4 months after co-inoculation), mycorrhizal plantlets had better growth with new root and shoot production than non-mycorrhizal plantlets (Fig. 2g, h). However, the magnitude of response varied with treatments. 123 – e 9.28 IBA (lM/l) 24 – – Table 2 Effect of auxins on root induction from regenerated shoots of Cymbidium bicolor F value5, 2.88 ± 0.74f,g,h b 28.00 ± 0.31 – – Growth hormone 2.56 ± 0.12g,h a 17.80 ± 0.20 – – Values represent mean ± SE of three repeated experiments each with 10 replications. Means in a column with the different letter (superscript) are significantly different according to DMRT (P \ 0.05) – 2.44 ± 0.15h,i c 8.84 – *** Significant at P \ 0.001 – 9.60 ± 0.40 1.13 2.26 4.52 9.24 13.76 2.00 ± 0.29 i 4.40 ± 0.24a,b 2.60 ± 0.24 h,i 4.60 ± 0.15a,b 3.00 ± 0.18 h,i 3.76 ± 0.10c,d g 4.20 ± 0.20 5.20 ± 0.20f 3.32 ± 0.21d,f 2.36 ± 0.16i,j 761.68*** 24.56*** Mycorrhizal association significantly enhanced the number of roots, root length and shoot number (Table 3; Fig. 2i). Ex vitro establishment of plantlets After 60 days, the cover was gradually loosened, thus dropping the humidity (65–70 %). The procedure adopted subsequently resulted in successful in vitro hardening of the plants (Fig. 2j). Within 2 weeks, the plants were completely acclimatized and survived in open containers. This resulted in 90 % survival compared to 50 % in control. Fungal colonization The root of C. biolor consisted of velamen, exodermis with passage cells, cortex, endodermis and vascular region. The fungal hyphae entered the roots through the root hairs (Fig. 4a) or well-developed multilayered velamen before entering into exodermal passage cells. Passage cells appeared as sunken or broken cells in the exodermis where the fungal hyphae aggregated (Fig. 4b). Pelotons were numerous in the cortical cells. The pelotons were irregularly distributed in the cortex and were more frequent in the outer cortex than in the inner cortex (Fig. 4c). In colonized cortical cells, the nucleus was displaced to the periphery of the cell (Fig. 4d, e). The hyphae penetrated the cortical cell wall and continuously spread inwards forming pelotons (Fig. 4f). Individual hyphae constituting the peloton could be easily differentiated during initial stages but was gradually lost with time indicating its slow lysis and digestion (Fig. 4g, h). Intact and lysed pelotons occurred simultaneously in different cortical cells (Table 4). Acta Physiol Plant (2013) 35:829–840 835 Fig. 3 Morphological characterization of Moniliopsis sp. (AC-01). a, bMoniliopsis sp. (AC-01) cultured on PDA medium (bar 1.0 cm). c, d Hyphae in short chains (bar 10 lm). e, f Monilioid cells (bar 20 lm) Discussion In vitro seed germination has been suggested as a suitable propagation method for conservation of orchids (Kauth et al. 2006; Stewart and Kane 2006). According to Hartman et al. (1997) specific endogenous growth promoting and inhibiting compounds are involved directly in control of seed development, dormancy and germination. To initiate seed germination, three conditions must be fulfilled, which includes seed viability, appropriate environmental conditions and overcoming primary dormancy. In the present study successful seed germination and protocorm formation to a great extent was influenced by the quality of basal medium. Based on the previous reports on the effectiveness of various basal media for asymbiotic seed culture of orchids, the four basal media were selected. Of the four media tested in the present study, LO medium showed significantly higher (96.6 %) percentage of seed germination after 56 days of culture initiation. Besides LO medium, other three media also supported moderate germination (89 % in KCM, 77. 5 % in MS medium and 62.7 % in KC); however, germination was delayed and the germinated seeds failed to differentiate into either PLBs or plantlets properly. The nutrient regime for orchid culture is species 123 836 Acta Physiol Plant (2013) 35:829–840 Table 3 Effect of Moniliopsis sp. on growth of Cymbidium bicolor Treatment stage Control Inoculated t value Protocorms Plant height (cm) 0.56 ± 0.05 2.20 ± 0.20 -9.698*** No. shoots 2.00 ± 0.31 4.20 ± 0.37 -4.491** Plant height (cm) 3.80 ± 0.37 5.40 ± 0.40 -5.715* No. shoots 3.40 ± 0.24 12.80 ± 0.37 No. new roots Length of root (cm) 4.00 ± 0.44 5.80 ± 0.58 11.00 ± 0.44 15.80 ± 0.37 Seedling -8.485*** -13.898 -4.811** Values represent mean ± SE of three repeated experiments each with 10 replications based on Two Paired Samples test at P \ 0.05 *, **, *** Significant at P \0.5, P \ 0.01, P \0.001 respectively specific and no single culture medium is universally applicable for all the orchid species. For example MS medium for Malaxix khasiana (Deb and Temjensangba 2006), Coelogyne suaveolens (Sungkumlong and Deb 2008) and Cymbidium aloifolium (Deb and Pongener 2011), P723 medium for Eulophia alta (Johnson et al. 2007) and New Dogashima medium for Calanthe tricarinata (Godo et al. 2010) were reportedly most suitable over other nutrient media. In C. bicolor, the percentage of germination varied with media. All the media used in the present study differed in their chemical compositions. Mineral salts in the media varied not only in their concentrations, but also in their available forms. Briefly, MS medium is highly enriched with both macro- and micro-elements, whereas KC and KCM medium contained low amounts of both macro- and micro-elements and lacked vitamins and amino acids. Conversely, LO medium is moderate in both macro- and micro-elements but is enriched with amino acids and vitamins, thus conditions are suspected to be responsible for enhancing seed germination which is in agreement with many workers (Sharma et al. 1991; Kauth et al. 2006; Stewart and Kane 2006; Roy et al. 2011). Mariat (1949) reported that vitamin B favoured germination and differentiation of Cattleya seedlings; thiamine, nicotinic acid and biotin were most effective for Cattleya hybrids. Pyridoxine with nicotinic acid and biotin favoured better germination of Orchis laxiflora seeds (Mead and Bulard 1979). These findings indicate the existence of species–medium specificity. Such species-specific medium for seed germination have also been reported by many workers (Arditti and Ernst 1993; Bhadra et al. 2002; Kauth et al. 2008; Roy et al. 2011). The percentage of seed germination recorded in the germination test was higher compared to those recorded in 123 tetrazolium test. This suggests that tetrazolium test is unreliable for assessing C. bicolor seed viability. This contrasts the results reported for hard-seeded orchids such as Cypripedium (Lauzer et al. 1994; Vujanovic et al. 2000), Eulophia alta (Johnson et al. 2007) and Satyrium nepalense (Mahendran and Narmatha Bai 2009) where tetrazolium staining grossly overestimated germinability. Differences between estimated viability and observed germinability in C. bicolor may result from less optimal pretreatment or staining methods. The observations of the present and other studies clearly exemplify the need for direct seed germination tests in orchids rather than tetrazolium test for asserting seed viability. In C. bicolor, LO medium was supplemented with cytokinins such as BAP, KIN and TDZ for multiple shoot induction, the media turned brown with time possibly indicating the phenolic exudation. Browning of the media coincided with the arrest of seedling development, so the seedlings were transferred to B5 medium. Excessive phenolic production is possibly due to increased polyphenol oxidase and catalase activity triggered by certain culture conditions, such as improperly balanced nutrients or the lack of required growth stimulating substance as suggested by Stoutamire (1974) and Harvais (1982). The success of rapid and direct shoot regeneration from protocorm explant opens an efficient way to mass propagate C. bicolor. The type and concentration of growth regulators play an important role during in vitro propagation of many orchid species (Arditti and Ernst 1993). In the present study protocorms developed multiple shoots directly on B5 medium supplemented with cytokinins. Protocorms can possess several centers of meristematic activity, but usually develop only a single shoot as observed in nature (Tatarenko and Vakhrameeva 1998), whereas, in vitro, two pathways of protocorm cloning have been revealed: the formation of a great number of shoot apices, with further endogenous production of adventitious roots and the formation of numerous secondary protocorms from epidermal cells of a single protocorm (Andronova et al. 2000). In C. bicolor, the protocorm produced multiple shoots directly on the medium fortified with different cytokinins individually. Among the cytokinins tested, BAP was found more effective for inducing multiple shoots (28.00 ± 0.31/explant). BAP was also found suitable for protocorm and shoot multiplication in Geodorum densiflorum (Sheelavantmath et al. 2000), Cymbidium pendulum (Pathak et al. 2001), Geoderum purpureum (Mohapatra and Rout 2005), Dendrobium tranparens (Sunitibala and Kishor 2009) and Eulophia nuda (Panwar et al. 2012), whereas TDZ was beneficial in Satyrium nepalense (Mahendran and Narmatha Bai 2009), Aerides vandarum 9 Vanda stangeana (Kishor and Sunitibala Devi 2009). Acta Physiol Plant (2013) 35:829–840 Fig. 4 Anatomical studies showing association of the fungus in the roots of Cymbidium bicolor. a The entry (arrow) of the fungal hyphae (H) through the root hair (RH). b Colonized exodermal passage cell— Note the colonized cell with a fungal hyphae (arrow). c Formation of Pelotons in the cortical cell. d Transverse section of myorrhizal root showing the cortical cells packed with pelotons. e Cortical cells with 837 Pelotons—the nucleus is pushed aside in the cell protoplasm (arrow). fArrow points to the hyphae passing through the cell wall. g Cortical cells isolated and stained with tryphan blue, showing pelotons (arrow). h Lysing pelotons. Nucleus is pushed aside in the cell protoplasm (arrow). P pelotons. C cortical cell, LP lysing pelotons, H hyphae, RH roots hairs 123 838 Acta Physiol Plant (2013) 35:829–840 Table 4 Frequency of colonization in the root section of Cymbidium bicolor Conclusion Root and mycorrhizal characteristics An efficient method for the in vitro germination of seeds and for the regeneration of a large number of plantlets from protocorms for C. bicolor has been described. We isolated Moniliopsis sp. from A. crispum. This fungal isolate was capable of enhancing the growth of C.bicolor protocorm and seedling. As shown here, seeds of C. bicolor could germinate and seedlings could develop without fungal symbionts. However, inoculation of C. bicolor seedling with Moniliopsis sp. significantly increased plant growth and their survival rate. Further experiments are now being carried out to assess the in vitro growth promoting ability of this fungal strain in other orchid taxa. Number of root hairs 7.00 ± 0.316 Number of passage cells 12.60 ± 0.510 Number of pelotons in cortical layer 84.80 ± 5.791 Number of digested Pelotons in cortical layer Total number of cortical cells 17.40 ± 1.029 180.00 ± 26.840 Values represent mean ± SE of three repeated experiments each with 10 replications based on one sample test at P \ 0.05 The phenomena of mycotropism and mycorrhiza are discussed by many workers in both terrestrial and epiphytic orchids (Chang and Chou 2007; Chang et al. 2007; Fang et al. 2008). In the present study, the fungal isolate Moniliopsis sp. played a very important role in the seedling development. The mycorrhizal seedlings developed several new and long roots and shoots compared to their nonmycorhizal conspecifics. The fungal hyphae formed tightly interwoven pelotons which are considered to be the most distinctive characteristic future of orchid mycorrhiza (Currah and Zelmer 1992). Thus, an appropriate symbiotic relationship has been established between the inoculated orchid mycorrhizal fungus and C. bicolor seedlings. The fungal entry into roots was through the root hair or the velamen cells and spread through the cortical cells as observed in C. sinense and C. goeringii (Fan et al. 1999; Wu et al. 2005). The results of the present study clearly showed that mycorrhizal fungi improved the development of C. bicolor protocorms and seedlings. In the present study we just demonstrated the growth promoting ability of the endophyte, but the real mechanism remains unknown and needs further investigation. Observation similar to the present study was made in Cymbidium kanran cv. Hook. where mycorrhizal fungus inoculation stimulated plant growth parameters like plant height, leaf and root development and fresh weight (Lee et al. 1998). In addition, uptake of N by mycorrhizal C. kanran roots was more than non-mycorrhizal roots. Some studies do indicate that orchid mycorrhizal fungi secrete plant hormones which have been shown to have strong effects in improving the growth of orchids (Liu et al. 2010). Moniliopsis sp. isolated from A. crispum was able to improve the growth of C. bicolor. This clearly shows the lack of host specificity of this fungal strain and its host benefit across species. This is in accordance with the observation where an Epulorhiza repens strain (UAMH9824) isolated from Spiranthes brevilabris was able to promote seedling development in other taxa (see Massey and Zettler 2007). It can be concluded that the fungal strain Moniliopsis sp. used in the present study clearly enhanced the growth of C. bicolor in vitro. 123 Author’s contribution G. Mahendran carried out the entire research work on tissue culture and mycorrhiza. V. Muniappan helped in designing the experiment pertaining to mycorrhiza. M. Ashwini carried out a part of the tissue culture work. T. 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