South African Journal of Botany 104 (2016) 76–81
Contents lists available at ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Symbiotic seed germination of an endangered epiphytic slipper orchid, Paphiopedilum villosum (Lindl.) Stein. from Thailand N. Khamchatra a, K.W. Dixon b, S. Tantiwiwat a, J. Piapukiew c a b c
Department of Botany, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand Science Directorate, Botanic Gardens and Parks Authority, Kings Park and Botanic Garden, West Perth, Western Australia 6005, Australia Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
a r t i c l e
i n f o
Article history: Received 30 June 2015 Received in revised form 12 October 2015 Accepted 2 November 2015 Available online xxxx Edited by: M Kulkarni Keywords: Orchid mycorrhiza Mycorrhizal compatibility Epiphytic orchid Symbiotic germination Endophyte
a b s t r a c t Paphiopedilum villosum (Lindl.) Stein is a native epiphytic slipper orchid in Thailand. This species is now being threatened and endangered. Propagation of this species is essential for conservation and reintroduction purposes. In this study, the propagation of P. villosum was achieved through the in vitro asymbiotic and symbiotic seed germination. Seeds of P. villosum sown on asymbiotic media, Murashige and Skoog (MS), Vacin and Went (VW) and Thomale GD (TH), did not germinate within 16 weeks. Seven different fungal strains were isolated from roots of this orchid species. The germination rate index (GRI) and the development rate index (DRI) of P. villosum seeds in treatments inoculated with fungal isolates PVCP01, PVCP05, and PVCP06 was significantly higher than uninoculated control treatments. Fungal isolate PVCP01 significantly increased the GRI and DRI of every stage of protocorm development, whereas fungal isolates PVCP05 and PVCP06 were only able to promote seed germination and protocorm development to stage 2. As for the wild orchid species, P. villosum, a compatible fungus is therefore required for promoting seed germination and protocorm development. Based on analysis of morphological characters and sequences of the nuclear ribosomal transcribed spacer (ITS), fungal isolates PVCP01, PVCP 05, and PVCP06 were identified as Tulasnella sp., Ceratobasidium sp., and Flavodon sp., respectively. The information obtained from this study will be used to propagate other threatened Thai orchids for conservation and reintroduction programs. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction The Orchidaceae has a symbiotic relationship with mycorrhizal fungi in order to permit seed germination, protocorm development, and seedling growth. Because of a lack of sufficient nutrient reserves in orchid seeds, orchids rely on compatible fungal partners to supply carbon sources and other nutrients until plants reach a level of autotrophy (Leake, 1994; Rasmussen, 1995; Steinfort et al., 2010). The specificity of the orchid–mycorrhiza interaction is an important key to determine chances of seedling establishment (Bidartondo and Read, 2008) although some orchids associate with more than one mycorrhizal fungal species and vice versa (Otero et al., 2004; Bonnardeaux et al., 2007; Stewart and Kane, 2007; Steinfort et al., 2010). Moreover, it has been found that different fungi might be required to progress plant development from germination until plants attain adulthood (Rasmussen, 2002; Steinfort et al., 2010). For ex situ propagation for conservation translocations, it is therefore necessary to understand the requirements for fungal endophytes from the germination through the critical stages of seedling establishment.
E-mail address:
[email protected] (J. Piapukiew).
http://dx.doi.org/10.1016/j.sajb.2015.11.012 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
Paphiopedilum villosum (Lindl.) Stein is a rare epiphytic or occasionally lithophytic orchid restricted to Assam India, Myanmar, Northern Thailand, Southern China, Laos, and Vietnam (Pfahl et al., 2015) flowering from December to April with a low natural seed set of 8% (Bänziger, 1996). Over collection of P. villosom coupled with low frequency of fruit set and limited evidence of prolific seed germination in the wild has resulted in this taxon being rare and threatened in the wild with all species of Paphiopedilum listed as Schedule 1 species under CITES provisions In vitro propagation of Paphiopedilum species is often considered difficult because seed germination, growth, and development depend on different factors such as capsule maturity, macro- and microelement composition, culture method, and often the addition of nonspecific growth factors (Arditti, 1967; Long et al., 2010; Hossain et al., 2013; Zeng et al., 2013). Although commercial axenic production of Paphiopedilum species has been achieved for most species (Nhut et al., 2005; Liao and Chen, 2006; Lee, 2007; Long et al., 2010; Zeng et al., 2006, 2010, 2012, 2013; Chen et al., 2015), symbiotic seed germination of Paphiopedilum species has not been reported yet this approach may be critical to ensure that plants used in translocations include a suitable mycobiont. The present study therefore aimed (1) to evaluate the potential of asymbiotic seed germination and protocorm development of P. villosum to provide a benchmark for assessing the performance of
N. Khamchatra et al. / South African Journal of Botany 104 (2016) 76–81
symbiotic procedures, (2) to isolate and identify root-associated fungi isolated from wild plants, and (3) to determine the ability of the fungi to promote seed germination and protocorm development. The results obtained from this study will be used to propagate orchids to aid future orchid conservation and reintroduction programs. 2. Material and methods 2.1. Plant material Whole plants of P. villosum (n = 5) were kindly provided by The Royal Conservation Paphilopedilum Projects, Chiangmai, Thailand, in 2008–2009 (Fig. 1a). All wild plant sources were collected from various forest sites in Chiangmai province. Mature undehisced capsules of P. villosum were collected from The Royal Conservation Paphilopedilum Projects after 10 months following cross-pollination by hand (Fig. 1b). The capsules were stored in paper envelopes and desiccated over silica gel for 3–4 days until dehiscence of capsules. Viability of seeds was determined within 7 days using tetrazolium test (Van Waes and Bebergh, 1986). Brown seeds from dehisced capsules were stored in sterile Eppendorf tubes at 4 °C until used in the seed germination experiment.
77
with a PCR profile of 94 °C for 5 min, followed by 38 cycles of denaturation at 94 °C for 1 min, annealing at 51 °C for 1 min and extension at 72 °C for 1 min, followed by 5 min of final extension at 72 °C. The PCR products were purified using the NucleoSpin® kit (Macherey-Nagel Inc., Easton, USA). Direct sequencing was performed on both stands of DNA using the PCR primers mentioned above by Macrogen, Inc. (Seoul, Korea). BLAST search (http://blast.ddbj.nig.ac.jp/top-e.html, Altschul et al., 1997) was performed on all sequences to determine their close known relatives. 2.4. Asymbiotic germination on basal media The influence of basal medium on seed germination and protocorm development was evaluated by sowing seeds on three basal media: (1) Murashige and Skoog (MS; Murashige and Skoog, 1962), (2) Vacin and Went (VW; Vacin and Went, 1949), and Thomale GD (TH; Thomale, 1954). For each treatment, approximately 100 surface sterilized seeds were cultured in a 120 ml culture bottle containing 20 ml of solidified medium. All experiments consisted of three independent replicates with nine culture bottles per replicate incubated at 25 °C. 2.5. Symbiotic seed germination
2.2. Fungal isolation The roots of P. villosum were rinsed in tap water to remove debris and cut in 1 cm segments. The segments were sterilized in 0.5% NaOCl for 5 min and finally rinsed in sterile distilled water three times. The surface-sterilized segments were transferred to a sterile Petri dish and immersed in sterile water. The segments were cut longitudinally and pelotons were removed (Fig. 1c) from cortical cells using a dissecting needle under a stereomicroscope (Nikon SMZ-2B). The individual pelotons were washed with five changes of sterile distilled water. Each peloton was then placed on a Petri plate containing 1/6 Nutrient Dextrose Yeast Extract agar (1/6NDY) supplemented with 100 μg/ml of streptomycin and 100 μg/ml tetracycline. Plates were incubated at 25 °C in the dark and observed periodically until fungal colonies developed. Fungal mycelia of each colony were sub-cultured onto Potato Dextrose Agar (PDA) plates. Pure cultures were stored on 1/5 PDA slants at 15 °C.
The effects of fungal isolates on promoting P. villosum symbiotic seed germination in vitro were evaluated using a modified method of Stewart and Kane (2006). Seeds were surface sterilized as described by Batty et al. (2001). Seeds in a filter paper pocket were immersed into two changes of sterile distilled water for 2 min, followed by 0.5% (v/v) NaOCl for 10 min and finally in three changes of sterile distilled water. Approximately 50–100 surface sterilized seeds were sown onto the surface of a one-eighth strip of a sterile Whatman no. 4. filter paper and placed onto media in Petri dish containing 20 ml of sterile Oat Meal Agar (OMA) (pH 6.0). The plates were inoculated with a 5-mmdiameter plug of each fungal inoculum taken from the actively growing hyphal edge 7 days after culturing on PDA. Uninoculated plates were used as a control. The number of replicates for each treatment was four. Petri dish plates were sealed with parafilm and stored in the dark at 25 °C for 16 weeks. Randomly sampled protocorms were examined microscopically to confirm the mycobiont structures under a compound light microscope.
2.3. Fungal identification 2.6. Symbiotic cultivation The fungal isolates were first identified using morphological characteristics and the methods outlined by Barnett and Hunter (1987), Zelmer and Currah (1995), Zelmer et al. (1996), and Currah et al. (1997). Molecular identification was based on ITS sequences. DNA was prepared from 7 to 10 day-old fresh culture and extracted with cetyltrimethylammonium brominde (CTAB) method as described by Zhou et al. (1999). The primers ITS1 and ITS4 (White et al., 1990) were used for ITS amplification. PCR amplification was performed in a total volume of 35 μl which included 100 ng genomic DNA, 1 × PCR master Mix (Fermentas, California, USA), and 100 nM of each primer. The amplification was carried out in a thermocycler (Takara, Japan)
The protocorms in stage 5 on OMA were randomly sampled for monitoring survival, growth, and developmental stage. The protocorms were exposed to an 8-h white light photoperiod provided by fluorescent lamps at 40 μmol m−2 s−1 for 2 weeks. Seedlings with two leaves were transferred to polycarbonate containers (650 mm3) containing 100 ml sterile OMA at the bottom covered with sterile silica sand. The containers were maintained at 25 ± 2 °C and 70% relative humidity for 4 months. The seedlings were transferred to new containers containing 100 ml sterile OMA covered with a (1:1:1, v:v:v) sterilized mixture of peat moss, vermiculite, and perlite. After 24 months, the
Fig. 1. a Flowering of Paphiopedilum villosum (Lindl.) Stein. as an epiphytic plant, bar = 1 cm. b Mature capsule of P. villosum and c pelotons in the cortical cells of extracted roots (arrow).
78
N. Khamchatra et al. / South African Journal of Botany 104 (2016) 76–81
seedlings were removed from the containers to plastic pots containing volcanic rocks, pumice stone, vermiculite, sand, coconut dust, and pine bark (0.5:0.5:1:1:1:2, v:v:v:v:v:v) for hardening and after that all plants were transferred to greenhouse conditions.
based on BLAST sequence similarity and the identities of the most closely matched sequences obtained by BLAST search are shown in Table 2. Six fungal isolates were Basidiomycota, while the remaining isolate PVCP09 was a member of Ascomycota.
2.7. Data collection
3.2. Asymbiotic seed germination
Seed germination and protocorm development for both the asymbiotic and symbiotic experiments were observed every 4 weeks under a stereomicroscope. The germination and the developmental stage were scored on a 1–5 incremental growth scale (Table 1; Stewart and Kane, 2007). Percentage of seed germination and protocorm development for each treatment was calculated by dividing the number of seeds in each developmental stage by the total number of viable seeds. Germination rate index (GRI) and developmental rate index (DRI) were determined as described in Papenfus et al. (2015):
The results of tetrazolium testing for seed viability prior to experimentation showed that P. villosum seeds were 37.6% viable. Seeds of P. villosum on all tested media did not germinate within 16 weeks (Table 3). The seed germination of P. villosum were continued to be observed. The result found that seeds germinated within week 20 and 28 after sowing on VW and MS media, respectively (data not shown). Seed development of P. villosum on both media was arrested at stage 2. No seed germination appeared on TH medium.
GRI ¼
G1 G2 Gx þ þ…þ 1 2 x:
where G1 is equal to the germination percentage × 100 at the first count after sowing; G2 is equal to the germination percentage × 100 at the second count after sowing, etc. DRI ¼
D1 D2 Dx þ þ…þ 1 2 x
where D1 is equal to the percentage of protocorms of a particular developmental stage (stage 2–5) × 100 at the first count after sowing; D2 is equal to the percentage of a particular developmental stage (stage 2–5) × 100 at the second count after sowing, etc. 2.8. Statistical analysis All experiments were established in a completely randomized design (CRD). The data were square root arcsine transformed prior to analysis to normalize variability. Analysis of variance (ANOVA) was performed with SPSS V16.0 statistical package (SPSS Inc., Chicago, USA) and the means compared by Duncan's multiple range test (P = 0.05).
3.3. Symbiotic seed germination and cultivation An effect of seven fungal isolates on seed germination and protocorm development of P. villosum was monitored and evaluated after sowing for 16 weeks as shown in Table 3. The GRI of P. villosum seeds in treatments inoculated with fungal isolates PVCP01, PVCP05, PVCP06, PVCP08, PVCP09, and nonioculated OMA treatment was significantly higher than other treatments. The GRI of treatments inoculated with fungal isolates PVCP04 and PVCP07 was not significantly different than asymbiotic treatments MS, VW, and TH. Both fungal isolates overgrew and degraded the seeds 30 days after sowing. Seeds inoculated with fungal isolate PVCP01 showed the highest GRI (28.36% per week). Furthermore, this fungal isolate supported an advanced protocorm developmental stage up to stage 5 with giving the highest DRI (0.59% per week). In addition, P. villosum seeds inoculated with fungal isolates PVCP06 significantly increased the DRI of protocorms at stage 2 as compared to those inoculated with isolate PVCP05. The seed development in treatments inoculated with fungal isolates PVCP05 and PVCP06 was then arrested at stage 2. The protocorm development of each stage was shown in Fig. 3a–g. All sampled seedlings survived and grew vigorously after transplanting (Fig. 3i–k). The seedlings from symbiotic seed germination were successfully acclimated to greenhouse conditions for 2 years after initial germination was achieved (Fig. 3l).
3. Results 4. Discussion 3.1. Fungal isolation and identification Seven endophytic fungal isolates were recovered from the roots of P. villosum. The fungal isolate PVCP01 and isolate PVCP05 were identified as Rhizoctonia-like fungi based on morphological and cultural characteristics. The fungal isolate PVCP01 on PDA had white colony characteristics, and spherical monilioid cells after 7 days of culture (Fig. 2a, c). The colony color of fungal isolate PVCP05 on PDA was creamy white. The ellipsoidal monilioid cells were observed after 7 days of culture (Fig. 2b, d). The ITS sequences of seven fungal isolates were compared with available sequences in the DNA Data Bank of Japan (DDBJ). Putative taxonomic affinities were assigned conservatively to the fungal isolates Table 1 Developmental stages of symbiotically cultured Paphiopedilum villosum seeds and protocorms (adapted from Stewart and Kane, 2007). Stage
Description
0 1 2 3 4 5
No germination, testa intact Embryo swollen (=germination) Continued embryo enlargement, testa ruptured, rhizoids present Appearance of protomeristem Emergence of first leaf Elongation of first leaf and further development
To conserve and reintroduce threatened and endangered orchid species, it is necessary to propagate these orchid species with a preference for symbiotic approaches. Symbiotic germination has become a popular and useful method for orchid seed propagation particularly where species are require for reintroduction programs (Stewart and Kane, 2006; Chutima et al., 2011; Fracchia et al., 2014). Nevertheless, there is no study reporting symbiotic seed germination of an endangered Paphiopedilum even though many of these taxa are critically threatened by over-exploitation and habitat loss. Thereby, this is a first report of in vitro symbiotic germination of the rare P. villosum. All fungal isolates were endophytic in roots of P. villosum with morphological and molecular characters indicating that fungal isolates PVCP01 and PVCP 05 (Rhizoctonia-like isolates) were a Tulasnella sp. and Ceratobasidium sp., respectively. These fungal genera are well known as mycorrhizal fungi and associated with a diverse range of terrestrial and epiphytic orchids, including Acianthus, Bipinnula, Diuris, Goodyera, Platanthera, Caladenia, Dendrobium, Cymbidium, and Paphiopedilum, (Warcup, 1973, 1981; Rasmussen, 2002; Athipunyakom et al., 2004; Nontachaiyapoom et al., 2010; Steinfort et al., 2010). Athipunyakom et al. (2004) revealed that many fungal species isolated from species of Paphiopedilum were members of Tulasnella. Additionally, Nontachaiyapoom et al. (2010) reported that Epulorhiza calendulina-like isolates, which are anamorphic states in the genus Tulasnella, were only
N. Khamchatra et al. / South African Journal of Botany 104 (2016) 76–81
79
Fig. 2. Cultural and morphological characters of fungal isolate PVCP01 and isolate PVCP05 on Potato Dextrose Agar at 7 days after culture. a Culture of isolate PVCP01, bar = 2 cm; b culture of isolate PVCP05, bar = 2 cm; c monilioid cells of isolate PVCP01, bar = 20 μm; and d monilioid cells of isolate PVCP05, bar = 20 μm.
found in the roots of Paphiopedilum. Based on the comparison of ITS sequences, fungal isolate PVCP04, PVCP06, PVCP07, PVCP08, and PVCP09 were identified as Rigidoporus, Flavodon, Antrodia, Ganoderma, and Valsa, respectively. Most of these isolates were a group of Basidiomycota except that isolate PVCP09 was a member of the Ascomycota. These fungal isolates have not been reported as associates of orchid roots, and though they were, endophytes may have represented casual, nonmycorrhizal endophytes. Some of these fungi have been previously reported as root rot fungi and to be capable of phytopathogenicity (Old et al., 1991; Sudirman et al., 1992; Paterson, 2007; Raghukumar et al., 2004). In addition, the results of symbiotic seed germination in this study showed that isolate PVCP06 could promote the germination of P. villosum to stage 2, while fungal isolate PVCP04 overgrew and parasitized seeds. However, it is difficult to designate the ecological roles of these isolates as the impacts on the seed may not reflect ecological competency in the adult plant. The results of asymbiotic seed germination of P. villosom showed no seed germination on MS, VW, and TH media within 16 weeks, whereas seeds on OMA medium were able to germinate (Table 3). These media failed to support protocorm development. This indicated that all tested media were not suitable for seed germination and protocorm development of this orchid species. The type of medium was one factor affecting seed germination and seedling development of Paphiopedilum. Most asymbiotic germination media contain similar compositions such as Table 2 Molecular identification of seven endophytic fungal isolated from wild Paphiopedilum villosum plants. Isolate
Accession no.
Identity (%)
BLAST search result (Accession no./ taxonomic affiliation)
PVCP01 PVCP04 PVCP05 PVCP06 PVCP07 PVCP08 PVCP09
AB971169 AB971170 AB971171 AB971172 AB971173 AB971174 AB971175
95 99 91 99 95 99 97
Tulasnella sp. (AY373281)/Tulasnellaceae Rigidoporus vinctus (HQ400710)/Polyporales Ceratobasidium sp. (HM117643)/Tulasnellaceae Flavodon flavus (JQ638521)/Polyporales Nigroporus vinosus (AB811859)/Polyporales Coriolopsis retropicta (KC867403)/Polyporales Valsa eugeniae (AY347344)/Diaporthales
sugar, mineral salts, and agars. Many studies have reported that nitrogen source and concentration can play an important role during in vitro asymbiotic orchid seed germination (Curtis, 1947; Malmgren, 1992; Kauth et al., 2006; Stewart and Kane, 2006; Johnson et al., 2007). It has been known that most Paphiopedilum species prefer a low salt medium for seed germination and protocorm development (Zeng et al., 2012; Chen et al., 2015). Long et al. (2010) found that the relatively low mineral salt and inorganic nitrogen concentration in VW medium promoted seed germination of P. villosum var. densissinum. In addition, Chen et al. (2015) demonstrated that high salt concentrations in the medium and extended exposure to residual NaCl reduced germination of P. spicerianum seeds because of damaging testa and embryo. Besides, several studies reveal that MS medium may inhibit germination of Paphiopedilum seeds because of its high nutrient content (Pierik et al., 1988; Chen et al., 2004; Ding et al., 2004; Long et al., 2010), whereas 1/2, 1/4, 1/5, and 1/8 MS have been demonstrated to be more suitable (Ding et al., 2004; Long et al., 2010; Zeng et al., 2012). TH medium have been reported to be usually used at an early stage of seedling development (Lucke, 1971; Ernst, 1974, 1975; Stimart and Ascher, 1981). However, the ideal medium for the seed germination of Paphiopedilum species is known to be species-specific (Zeng et al., 2012; Chen et al., 2015). In symbiotic seed germination in the present study, the seeds in treatments inoculated with fungal isolate PVCP01 developed much faster than other treatments (Table 3). The treatment inoculated with this fungal isolate also increased the GRI and DRI of every developmental stage (stage 2–5; Table 3). These results indicated that the fungal isolate PVCP01 promoted germination and development of P. villosum seeds. Furthermore, this fungal isolate also supported growth, development, and survival of P. villosum seedlings after transplanting in greenhouse condition (Fig. 3k, l). As stated previously, compatible fungi must be capable of promoting seed germination and development to stage 5 and beyond (Bonnardeaux et al., 2007; Nontachaiyapoom et al., 2011). It is well known that mycorrhizal fungi can provide suitable carbon sources, which are essential to seed germination and protocorm development (Tan et al., 2014). Therefore, it is necessary to examine the ability of some single fungus for stimulating in vitro seed
80
N. Khamchatra et al. / South African Journal of Botany 104 (2016) 76–81
Table 3 Effect of basal media and seven fungal isolates on germination and development of P. villosum seeds for 16 weeks after sowing. Developmental stages
Stage 1
Stage 2
Stage 3
Stage 4
Stage 5
Treatments
GRI (% per week)
DRI (% per week)
DRI (% per week)
DRI (% per week)
DRI (% per week)
MS VW TH OMA PVCP01 PVCP04 PVCP05 PVCP06 PVCP07 PVCP08 PVCP09
0.f 0f 0f 0f 28.36 ± 0.94 a 0f 5.36 ± 0.29 c 7.51 ± 0.51 b 0f 1.06 ± 0.03 d 0.86 ± 0.06 e
0d 0d 0d 0d 10.33 ± 0.90 a 0d 0.21 ± 0.03 c 0.35 ± 0.04 b 0d 0d 0d
0b 0b 0b 0b 3.06 ± 0.19 a 0b 0b 0b 0b 0b 0b
0b 0b 0b 0b 1.00 ± 0.18 a 0b 0b 0b 0b 0b 0b
0b 0b 0b 0b 0.59 ± 0.05 a 0b 0b 0b 0b 0b 0b
Means ± SD followed by the same letter along columns are not significantly different by Duncan's multiple range test at 0.05% probability level.
germination or to demonstrate the symbiotic relationships between two organisms. The results of this study indicate that fungal isolate PVCP01 is compatible with P. villosum and represents an isolate that has ecological value for the species. Although Athipunyakom et al. (2004) and Nontachaiyapoom et al. (2010) isolated many fungal strains from roots of P. villosum, there is no study reporting successful in vitro symbiotic propagation of this orchid species until now. One reason for this lack of symbiotic success in P. villosum is that seed germination and further development of P. villosum need high degree of mycobiont specificity under in vitro symbiotic seed germination conditions and
prolonged culture periods. Furthermore, Zettler (1997) found that if an orchid relies on a compatible mycorrhizal fungus for germination, the loss of that fungus in situ may underpin the failure of the plant to establish natural conditions during a reintroduction program, for example. An additional advantage of symbiotic propagation of orchids is the resulting seedlings can serve as plant material and fungal inoculum for conservation activities (Batty et al., 2006; Johnson et al., 2007). Resolving an efficacious mycobiont for P. villosum is a promising step forward in continuing efforts to develop reintroduction and conservation procedures for this rare and threatened orchid species, with the
Fig. 3. Symbiotic seed germination and protocorm developmental stages of P. villosum inoculated with fungal isolate PVCP 01 cultured on Oat Meal Agar. a Stage 0 (arrow) no germination, testa intact, bar = 1 mm; b stage 1 (arrow) embryo swollen (=germination), bar = 1 mm; c stage 2 (arrow) continued embryo enlargement, testa ruptured, bar = 0.8 mm; d stage 3 (arrow) appearance of protomeristem, bar = 1 mm; e stage 4 (arrow) emergence of first leaf, bar = 1 mm; f stage 5 (arrow) elongation of first leaf and further enlargement of protocorm, bar = 7.5 mm. g Colonization of fungal isolate PVCP01 (arrow) in protocorm cells, bar = 0.3 mm. h Protocorms at stage 5 before subculturing, bar = 4 mm. i Seedling growth on OMA, bar = 1.0 cm; j seedling growth on sand supplemented with OMA at 4 months after transferring, bar = 1 cm; k seedling growth following transfer to soil mixtures (vermiculite: peat mosses: sand; 1:1:1 by volume), bar = 2 cm. l 2 year old symbiotic P. villosum seedlings for transplanting into natural conditions, bar = 5 cm.
N. Khamchatra et al. / South African Journal of Botany 104 (2016) 76–81
techniques employed here being relevant to other endangered and threatened wild orchid species. Further study should focus on improving the efficiency of P. villosum asymbiotic seed germination such as refining media composition, ex vitro seed germination, acclimatization, and in situ establishment methods to success in rare orchid conservation and recruitment programs. Acknowledgments Support for this work was granted by the Agricultural Research Development Agency (Public Organization), Bangkok, Thailand (CRP5205010090). We would like to thank The Royal Conservation Paphilopedilum Projects for providing plant materials. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–3402. Arditti, J., 1967. Factors affecting the germination of orchid seeds. Botanical Review 33, 1–97. Athipunyakom, P., Manoch, L., Piluek, C., 2004. Isolation and identification of mycorrhizal fungi from eleven terrestrial orchid. Kasetsart Journal (Natural Science) 38, 216–228. Barnett, H.L., Hunter, B.B., 1987. Illustrated Genera of Imperfect Fungi. fourth ed. Macmillan, New York. Bänziger, H., 1996. The mesmerizing wart: the pollination strategy of epiphytic lady slipper orchid Paphiopedilum villosum (Lindl.) Stein (Orchidaceae). Botanical Journal of the Linnean Society 121, 59–90. Batty, A.L., Brundrett, M.C., Dixon, K.W., Sivasithamparam, K., 2006. In situ symbiotic seed germination and propagation of terrestial orchid seedlings for establishment at field sites. Australian Journal of Botany 54, 375–381. Batty, A.l., Dixon, K.W., Brundrett, M., Sivasithamparam, K., 2001. Constraints to symbiotic germination of terrestial orchid seed in a Mediteranean bushland. New Phytologist 152, 511–520. Bidartondo, M.I., Read, D.J., 2008. Fungal specificity bottlenecks during orchid germination and development. Molecular Ecology 17, 3707–3716. Bonnardeaux, Y., Brundrett, M., Batty, A., Dixon, K.W., Koch, J., Sivasithamparam, K., 2007. Diversity of mycorrhizal fungi of terrestial orchids: compatibility webs, brief encounters, lasting relationships and invasions. Mycological Research 111, 51–61. Chen, Y., Googale, U.M., Fan, X.L., Gao, J.Y., 2015. Asymbiotic seed germination and in vitro seedling development of Paphiopedilum spicerianum: an orchid with extremely small population in China. Global Ecology and Conservation 3, 367–378. Chen, Y., Ye, X.L., Liang, C.Y., Duan, J., 2004. Seed germination in vitro of Paphiopedilum armeniacum and P. micranthum. Acta Horticulturae Sinica 31, 540–542. Chutima, R., Dell, B., Vessabutr, S., Bussaban, B., Lumyong, S., 2011. Endophytic fungi from Pecteilis susannae (L.) Rafin (Orchidaceae), a threatened terrestrial orchid in Thailand. Mycorrhiza 21, 221–229. Currah, R.S., Zettler, I.W., McInnis, T.M., 1997. Epulorhiza inquilina sp. nov. from Plantanthera (Orchidaceae) and a key to Epulorhiza species. Mycotaxon 61, 335–342. Curtis, J.T., 1947. Studies on the nitrogen nutrition of orchid embryos. American Orchid Society Bulletin 16, 654–660. Ding, C.C., Wu, H., Liu, F.Y., 2004. Factors affecting the Germination of Paphilopedilum armeniacum. Acta Botanica Yunnanica 26, 673–677. Ernst, R., 1974. The use of activated charcoal in asymbiotic seedlings culture of Paphiopedilum. American Orchid Society Bulletin 43, 35–38. Ernst, R., 1975. Studies in asymbiotic seedlings culture of orchids. American Orchid Society Bulletin 444, 12–18. Fracchia, S., Aranda-Rickert, A., Flachsland, E., Terada, G., Sede, S., 2014. Mycorrhizal compatibility and symbiotic reproduction of Gavilea australis, an endangered terrestrial orchid from south Patagonia. Mycorrhiza 24, 627–634. Hossain, M.M., Kant, R., Van, P.T., Winarto, B., Zeng, S.J., Teixeira da Silva, J.A., 2013. The application of biotechnology to orchid. Critical Reviews in Plant Sciences 32, 69–139. Kauth, P.J., Vendrame, W.A., Kane, M.E., 2006. In vitro seed culture and seedling development of Calopogon tuberosus. Plant Cell Tissue and Organ Culture 85, 91–102. Johnson, T.R., Stewart, S.L., Dutra, D., Kane, M.E., Richardson, L., 2007. Asymbiotic and symbiotic seed germination of Eulophia alta (Orchidaceae)-preliminary evidence for the symbiotic culture advantage. Plant Cell Tissue and Organ Culture 90, 313–323. Leake, J.R., 1994. Transley review no. 69. The biology of mycoterotrophic (‘saprophytic’) plants. New Phytologist 127, 171–216. Lee, Y.L., 2007. The asymbiotic seed germination of six Paphiopedilum species in relation to the time of seed collection and seed pretreatment. Acta Horticulturae 755, 381–385. Liao, Y.Z., Chen, J.J., 2006. Asymbiotic seed germination of Paphiopedilum. Paphiopedilum in Taiwan IV. Taiwan Paphiopedilum Society, pp. 11–14. Long, B., Niemiera, A.X., Cheng, Z.Y., Long, C.L., 2010. In vitro propagation of four threathened Paphiopedilum species (Orchidaceae). Plant Cell, Tissue and Organ Culture 101, 151–162. Lucke, R., 1971. The effect of biotin on sowing of Paphiopedilum. American Orchid Society Bulletin 40, 24–26. Malmgren, S., 1992. Large-scale asymbiotic propagation of Cypripedium calceolus-plant physiology from a surgeon's point of view. Botanic Gardens Micropropagation News 1, 59–63.
81
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497. Nhut, D.T., Trang, P.T.T., Vu, N.H., Thuy, D.T.T., Van Khiem, D., Van Binh, N., Van, K.T.T., 2005. A wounding method and liquid culture in Paphiopedilum delenatii propagation. Propagation of Ornamental Plants 5, 158–163. Nontachaiyapoom, S., Sasirat, S., Manoch, L., 2010. Isolation and identification of Rhizoctonia-like fungi from roots of three orchid gemera, Paphiopedlum, Dendrobrium, and Cymbidium, collected in Chaing Rai and Chiang Mai provinces of Thailand. Mycorrhiza 20, 456–471. Nontachaiyapoom, S., Sasirat, S., Manoch, L., 2011. Symbiotic seed germination of Grammatophyllum speciosum Blume. and Dendrobium draconis Rchb. f., native orchids of Thailand. Scientia Horticulturae 130, 303–308. Old, K.M., Yuan, Z.Q., Kobayashi, T., 1991. A Valsa teleomorph for Cytospora eucalypticola. Mycological Research 95, 1253–1256. Otero, J.T., Ackerman, J.D., Bayman, P., 2004. Differences in mycorrhizal preferences between two tropical orchids. Molecular Ecology 13, 2393–2404. Papenfus, H.B., Naidoo, D., Posta, M., Finnie, J.F., Van Staden, J., 2015. The effects of smoke derivatives on in vitro seed germination and development of the leopard orchid Ansellia africana. Plant Biology http://dx.doi.org/10.1111/plb.12374. Paterson, R.R.M., 2007. Ganoderma disease of oil palm—a white rot perspective necessary for integrated control. Crop Protection 26, 1369–1376. Pfahl, J., Taylor, S., Kirsch, C., Alford, D., 2015. Internet orchid species photo encyclopedia. http://www.orchidspecies.com/paphvillosum.htm. Pierik, R.L.M., Sprenkels, P.A., Vanderharst, B., Vandermeys, Q.G., 1988. Seed germination and futher development of plantlets of Paphiopedilum ciliolare pfitz in vitro. Scientia Horticulturae 34, 139–153. Raghukumar, C., Mohandass, C., Kamat, S., Shailaja, M.S., 2004. Simultaneous detoxification and decolorization of molasses spent wash by the immobilized white-rot fungus Flavodonflavus isolated from a marine habitat. Mycological Research 35, 197–202. Rasmussen, H.N., 1995. Terrestial Orchid: From Seed to Mycotrophic Plant. Cambridge University Press, Cambridge. Rasmussen, H.N., 2002. Recent developments in the study of orchid mycorrhiza. Plant and Soil 244, 149–163. Steinfort, U., Verdugo, G., Besoain, X., Cisternas, M.A., 2010. Mycorrhizal association and symbiotic germination of the terrestrial orchid Bipinnula fimbriata (Poepp.) Johnst (Orchidaceae). Flora 205, 811–817. Stewart, S., Kane, M., 2006. Symbiotic seed germination of Habenaria macroceratitis (Orchidaceae), a rare Florida terestial orchid. Plant Cell Tissue and Organ Culture 86, 159–167. Stewart, S., Kane, M., 2007. Symbiotic seed germination and evidance for in vitro mycobiont specificity in Spiranthes brevilabris (orchidaceae) and its implications for species-level conservation. In Vitro Cellular & Developmental Biology 43, 178–186. Stimart, D.P., Ascher, P.D., 1981. In vitro germination of Paphiopedilum seed on a completely defined medium. Scientia Horticulturae 14, 165–170. Sudirman, L.I., Housseini, A.I.I., Febvre, G.L., Kiffer, E., Botton, B., 1992. Screening of some basidiomycetes for biocontrol of Rigidoporus lignosus, a parasite of the rubber tree Hevea brasiliensis. Mycological Research 96, 621–625. Tan, X.M., Wang, C.L., Chen, X.M., Zhou, Y.Q., Wang, Y.Q., Luo, A.X., Liu, Z.H., Guo, S.X., 2014. In vitro seed germination and seedling growth of endangered epiphytic orchid, Dendrobium offcinale, endemic to China using mycorrhizal fungi (Tulasnella sp). Scientia Horticulturae 165, 62–68. Thomale, H., 1954. Die Orchidideen. Einfuhrung in die Kultur und Vermehrung tropischen und einheimischen Orchideen, second ed. Eugen Ulmer, Stuttgart, Germany. Vacin, E., Went, F.W., 1949. Some pH changes in nutrient solutions. Botanical Gazette 110, 605–613. Van Waes, J.M., Bebergh, P.C., 1986. Adaptation of the tetrazolium method for testing the seed viability and scanning electron microscopy study of some Western European orchids. Physiologia Plantarum 66, 435–442. Warcup, J.H., 1973. Symbiotic germination of some Australian terrestial orchids. New Phytologist 72, 41–46. Warcup, J.H., 1981. The mycorrhizal relationships of Australian orchids. New Phytologist 87, 371–381. White, T.J., Bruns, T., Lee, S., Taylor., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, pp. 315–322. Zelmer, C.D., Currah, R.S., 1995. Ceratorhiza pernacatena and Epulorhiza calendulina spp. nov: mycorrhizal fungi of terrestrial orchids. Canadian Journal of Botany 73, 15 (1981-1995). Zelmer, C.D., Cuthbertson, L., Currah, R.S., 1996. Fungi associated with terrestial orchid mycorrhizas, seeds and protocorms. Mycoscience 37, 439–448. Zeng, S.J., Chen, Z.L., Duan, J., 2006. Asepsis sowing and in vitro propagation of Paphiopedium hirsutissimum Pfitz. Plant Physiology Communication 42, 247. Zeng, S.J., Chen, Z.L., Wu, K.L., Zhang, J.X., Duan, J., 2010. In vitro propagation of Paphiopedilum henryanum Bream. Plant Physiology Communications 46, 471–472. Zeng, S.J., Wu, K.L., Teixeira da Silva, J.A., Zhang, J.X., Chen, Z.L., Xia, N.H., Duan, J., 2012. Asymbiotic seed germination, seedling development and reintroduction of Paphiopedilum wardii Sumerh., an endangered terrestrial orchid. Scientia Horticulturae 138, 198–209. Zeng, S.J., Wang, J., Wu, K., Teixeira da Silva, J.A., Zhang, J., Duan, J., 2013. In vitro propagation of Paphiopedilum hangianum Perner & Gruss. Scientia Horticulturae 151, 147–156. Zettler, L.W., 1997. Orchid-fungal symbiosis and its value in conservation vol. 13. McIlvainea, pp. 439–4448. Zhou, Z., Miwa, M., Hogetsu, T., 1999. Analysis of genetic structure of a Suillus grevillei population in a Larix kaempferi stand by polymorphism of inter-simple sequence repeat (ISSR). New Phytologist 144, 55–63.
