In vitro Symbiotic Seed Germination of formosum Roxb. ex Lindl.

Na-monrug Khamchatra1/2/ Somsak Apisitwanich4/ Kongkanda Chayamarit3/ Sureeya Tantiwiwat1/

ABSTRACT Most studies on orchid symbiotic seed germination involve with terrestrial and temperate orchids. There are few reports on association between epiphytic or tropical orchid species and symbiotic seed germination. This study was to determine the effect of mycorrhizal fungi on Dendrobium formosum symbiotic seed germination in vitro. Five mycorrhizal fungi isolated from D. formosum roots, were identified as genera Ceratobasidium (DFRN02), Cylindrocarpon (DFRN01 and DFRN03) and Epacris (DFRN04 and DFRN05). All fungal species were cultured into D. formosum seeds which were collected from provinces of Ranong and Chumphon in 2011-2012.,whilst on group of seeds were treated without fungal (control). After 3 months sowing, seeds of all treatments germinated over 60%. The germination (%) of DFRN02 treatment was significantly higher than other groups after 4 months sowing, followed by treatments of DFRN04, DFRN05 and DFRN01 respectively. The results indicates that Ceratobasidium sp. (DFRN02) can be a potential mycorrhizal fungus for D. formosum symbiotic seed germination and therefore for the conservation of this threatened orchid.

Key words: Dendrobium formosum, epiphytic orchid, mycorrhiza, symbiotic seed germination

1/ Department of Botany, Faculty of Science, Kasetsart University, Bangkok 10900, . 2/ Department of Biology, Faculty of Science and Technology, Rambhai Barni Rajabhat University, Chanthaburi 22000, Thailand. 3/ Department of National Parks, Wildlife and , Phahon Yothin Rd., Chatuchak, Bangkok 10900, Thailand. 4/ Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.

Thai Agricultural Research Journal Vol. 33 No. 2 May - August 2015 205 INTRODUCTION seed germination protocol to germinate The minute seeds of orchids are seeds of the rare subtropical terrestrial capable of dispersing across geographical orchid, Habenaria macroceratitis, was and ecological barriers, but contain very described, and the seed germination little stored energy to support germination percent was highest when seeds were (Arditti and Ghani, 2000). Orchid seeds sown with a fungal mycobiont, Epulorhiza depend on the energy and nutrients sp. (Stewart and Kane, 2006). The seed of supplied by a mycorrhizal fungus to several Australian temperate terrestrial germinate under natural conditions orchid taxa, Caladenia arenicola and (Rasmussen, 1995). Although orchid Pterostylis sanguinea, germinated best seeds were cultured to maturity using when seeds were sown with mycorrhizal asymbiotic techniques, asymbiotically fungi, and the seedling survival of grown seedlings rarely survive after being improved when actively growing symbiotic transferred to nature despite growing well seedlings were transferred to natural in vitro, besides the survival rate of orchid habitats during the growing season (Batty seedlings from symbiotic sowing was et al., 2006). Athipunyakom et al. (2004) higher than from asymbiotic sowing (Batty found that Spathoglottis plicata seeds, et al., 2006). The survival of orchids in inoculated with Epulorhiza repens and managed or restored habitats may require Rhizoctonia globularis isolated from 11 the presence of appropriate mycobionts terrestrial Thai orchid species, showed the to support development and initiation of leaves 60 days after sowing subsequent seedling recruitment (Zettler, seed. Seed germination and protocorm 1997). This is supported by well development of the threatened terrestrial documented cases where orchids have orchid, Pecteilis susannae (L.) Rafin were developed with fungi cultured on organic evaluated up to 70 days after sowing, and additives in vitro, or on organic debris the percent of symbiotic seed germination (Batty et al., 2001). was highest when seeds were cultured Several cases have been documented with Epulorhiza sp. (Chutima et al., 2011). for the transplant success from symbiotically Dendrobium is the second largest genus germinated orchids. An efficient symbiotic in the family . Dendrobium

206 วารสารวิชาการเกษตร ปีที่ 33 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2558 formosum Roxb. ex Lindl. is anendemic MATERIALS AND METHODS epiphytic wild orchid species in Southeast Plant materials Asia, widely distributed in , Whole plants of D. formosum Thailand, , and (Nanakorn (Figure 1a) were collected from anatural and Indharamusika, 1998). D. formosum is habitat near Ngao National Park located a species in the Convention on at Ranong (N.Khamchatra 11, N. International Trade in Endangered Species Khamchatra 12 and N. Khamchatra 13) of Wild Fauna and Flora (CITES) appendix and ChumphonProvinces (N. Khamchatra II listed plants (Convention on International 14 and N. Khamchatra 15), Thailand in Trade in Endangered Species of Wild 2011-2012. Nine to ten monthold D. Fauna and Flora, 2014), because they are formosum orchid capsules in yellow green large beautiful and fragrant flower and color were collected and kept in fragrant which make them worthy desiccated plastics boxes and stored in subjects for cultivation and hobbyists. the dark place for about one week at Unfortunately, the species has become room temperature. Afterward, the orchid threatened with extinction due to the loss seeds were transferred to sealed glass of its natural habitat as well as over- bottles before being stored at cold (4 ํ°C) harvesting for sale in illegal trade. The in dark place until use within 6 months. knowledge about propagation is needed Standard methods for collection for the conservation of this species. and preservation were used following Jain However, little is known about their and Rao (1977). The voucher specimens association with mycorrhiza fungi and were deposited at Plant Genetic their role in germination of D. formosum. Conservation Project Herbarium, Faculty Thus the objective of this study of Science and technology, Rambhai was to investigate the association Barni Rajabhat University, Chanthaburi between orchid mycorrhizal fungal and D. Province, Thailand. Flowers were fomosum symbiotic seed germination preserved in alcohol. Moreover, live (invitro) using mycobionts isolated from D. specimens are being maintained at the formosum in Thailand. first author’s garden.

Thai Agricultural Research Journal Vol. 33 No. 2 May - August 2015 207 Fungal isolation kept on 1/5 PDA slants and also placed The roots of D. formosum (Figure in sterile distilled water at 15 ํ°C for further 1b) were rinsed in tap water to remove use and 20% glycerol at -20° ํC for long- debris and cut in 1 cm segments. The term preservation. segments were sterilized in 0.5 % NaOCl for 5 min. and finally rinsed in sterile Morphological characterization of distilled water three times. The surface- isolates sterilized segments were transferred to a The 5 fungal isolates were first sterile Petri dish and immersed in sterile identified using morphological characteristics, water. The segments were cut longitudinally, according to the methods outlined by and pelotons (Figure 1c) were removed Robert (1999) and Athipunyakom etal. from cortical cells using a dissecting (2004). For determination of culture needle under a stereomicroscope (Nikon characteristics, all isolates were cultured SMZ-2B). The individual pelotons were on PDA and Oat Meal Agar (OMA) at 25 +± that opaque, circular-oval and submersible 2 ํ°C in the dark. Radial growth of the isolates were washed in five changes of sterile was measured 3 and 6 days after inoculation. distilled water. Each peloton (Figure 1d) Cultural morphology was recorded at first was then placed on a Petri plate and fourth week. The characteristics of containing 1/6 Nutrient Dextrose Yeast fungal isolates were recorded as color and Extract agar (1/6NDY) supplemented with growth of the colony, concentric rings 100 mg/ml of streptomycin and 100 mg/ formation and aerial hyphae, shape and ml tetracycline. Plates were incubated at size of the sclerotia. Hyphal and monilioid 25°C in the dark, and observed periodically length and width were measured. for fungal colonies development. Fungal Lactophenol cotton blue was used as a mycelia of each colony were sub-cultured staining agent for observing hyphal 2-3 times onto another fresh Potato characteristics and Safranin O-KOH was Dextrose Agar (PDA) plates to be purified used for staining the fungal nuclei. The and assigned the accession number, hyphal characteristics and the fungal nuclei starting with DFRN01, DFRN02, DFRN03, were examined and photographed under a DFRN04 and DFRN05.Pure cultures were Nikon Eclipse E400 microscope.

208 วารสารวิชาการเกษตร ปีที่ 33 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2558 Molecular analysis 1:10,00 dilution of TBE buffer and Molecular identification was based visualized by ultraviolet light on internal transcripbed spacer (ITS) transillumination. PCR products were sequences. All isolates were grown in 20 purified using the NucleoSpin® mL Potato Dextrose Broth (PDB) medium (Macherey-Nagel Inc., Easton, USA). in the dark at room temperature for 7 to Sequencing was performed on 23 ABI 10 days. A 50-100 mg mycelia aliquot 3730 XLs by using Big Dye Terminator was harvested from each culture and V3.1 Cycle Sequencing Kit (Macrogen, DNA was extracted with cetyltrimethy Inc., Korea). Each PCR template was lammonium bromide (CTAB) method as sequenced in both direction using either described by Zhou et al. (1999). forward (ITS1) or reverse (ITS4) primers. The primers ITS1 (5´-TCCGTAGG The sequenced were manually edited for TGAACCTGCGG-3´) and ITS4 (5´- ambigucus base with sequencer v. 4.0 TCCTCCGCTTATTG ATATGC-3´) (White et (Gene Codes Corporations). In order to al., 1990) were used for ITS amplification. determine the identity of these ITS Polymerase chain reaction (PCR) sequences, the “megablast” searches technique was used to amplify the ITS were performed on the U.S. National sequences according to the Thermo Center for Biotechnology Information Scientific PCR master mix kit (Fermentas, (NCBI: blast.ncbi.nlm.nih.sov). Pairwise California, USA). The amplification was sequence comparisons were expressed carried out in a thermocycler (Takara, as the percentage of the total number of Japan) with a PCR condition of 94 ํC nucleotide differences divided by total for 5 min, followed by 38 cycles of number of position sequence similarity of denaturation at 94 ํC for 1 min, annealing 95% or greater was considered. at 51 ํ°C for 1 min and extension at Symbiotic seed germination 72 ํ°C for 1 min, followed by 5 min. of final The effects of isolates on promoting extension at 72 °ํC. PCR products were D. formosum in vitro symbiotic seed separated by 1.5% agarose gel electrophoresis, germination were evaluated using a stained with SYBR Safe (Molecular method of Stewart and Kane (2006) with Probes, Eugene, OR) DNA gel stain, i.e. modification. Seed were surface sterilized

Thai Agricultural Research Journal Vol. 33 No. 2 May - August 2015 209 as described by Batty et al. (2001). light microscope. Percentages of seed Seeds in a filtered paper pocketwere germination were based on viable seeds immersed in distilled water with tween-20 using dissecting microscopy and protocorm for 2 min twice, followed by 0.5% NaOCl development were calculated by dividing for 10 min and finally rinsed 3 times in the number of seed in each germination sterile distilled water. Approximately 150- and development stage by the total 200 surface sterilized seeds were sown number of viable seeds in the sample onto the surface of a one-eighth of a (Arditii and Ghani, 2000). The data were sterile Whatman No.4 filtered paper and analyzed using Microsoft Office Excel placed in Petri dish containing 20 mL of program for one-way analysis of variance sterile Oat Meal Agar (OMA) at pH 6.0. A (ANOVA), and means were compared by 5- mm diameter plug of each fungal Duncan’s Multiple Range test inoculum, was taken from the actively growing hyphal edge 7 days after RESULTS AND DISCUSSION culturing on PDA, were placed at the Fungal isolation and identification middle of sown seeds plates. Uninoculated Intense colonization of peletons plates were used as control. Four was found in root of D. formosum (Figure replications were used for 6 treatments. 1). Intact or degraded pelotons were All 24 culture plates were sealed with observed in approximately 30-40% of root parafilm and then kept at 25 °ํC in the cortical cells with brownish color. dark for 4 months. Seed germination and Five fungal endophytic fungi isolates protocorm development were monitored were extracted from D. formosum roots. monthly under stereomicroscope and Based on morphology and molecular returned to the dark conditions. characteristics (Table 2), the fungal Developmental stages were recorded and isolates were divided into 2 groups. scored on a scale of 0-5 (adapted from Group I was the isolate DFRN02 which Arditii and Ghani, 2000; Table 1). showed resemblance to Rhizoctonia-like Protocorms were randomly sampled and orchid endophyte. The colony color of microscopically examined to confirm the isolate DFRN02 on PDA was creamy mycobiont structures under a compound white. The ellipsoidal monilioid cells were

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Figure 1 Dendrobiumformosum and peloton colonization: (a) Flower; (b) Root; (c) 15 PelotonsFigure in cortical 1Dendrobiumformosum cells; (d) Single and peloton peloton. colonization: (a) Flower; (b) Root; (c) Pelotons in cortical cells; (d) Single peloton.

Figure 2 Fungal colonies on PDA and morphological characteristics: (a) Isolate DFRN02 Figure 2 Fungal colonies on PDA and morphological characteristics: (a) Isolate DFRN02 colony on colony on PDA at 14 days; (b) Isolate DFRN02 colony on PDA at 7 days; (c) Right-angle PDA at 14 days; (b) Isolate DFRN02 colony on PDA at 7 days; (c) Right-angle branching hyphae; (d) branching hyphae; (d) Cluster of monilioid cells. Cluster of monilioid cells.

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Figure 3 SymbioticFigure 3Symbiotic seed seed germinationgermination and protocorm and development protocorm of D . developmentformosum inoculated with of D. formosum inoculated withCeratobasidium Ceratobasidium isolated fungi (DFRN02) isolated cultured fungi on OM (DFRN02)A: (a) control (uninoculated cultured seeds); on OMA: (b) (a) control (uninoculatedFungal seeds); colonization (b) of FungalCeratobasidium colonization sp. in protocorm; of (c) CeratobasidiumStage 2: continued embryo sp. enlargement, in protocorm; (c) Stage 2: continuedtesta ruptured; embryo (d) Stage 3: appearance enlargement, of protomeristem. testa ruptured; (d) Stage 3: appearance of protomeristem.

observed after 7 days (Figure 2). isolates were Group II consisting of 4

According to the BLAST search, isolate isolates, identified as Cylindrocarpon DFRN02 was identified as genus Gene Bank code: accession number

Ceratobasidium Gene Bank code: FJ605259 (Vaz et al., 2008), isolates accession number GU937735 (Jiang et al., DFRN01 and DFRN03 and Epacris Gene 2010). These fungal genera are well Bank code: accession number AY268191 known as mycorrhizal fungi and (Williams et al., 2003), isolates DFRN04 associated with a diverse range of and DFRN05 (Table 2). In this study, terrestrial and epiphytic orchids, including endophytic fungal were found as Acianthus R.Br., Bipinnula Comm. Ex Cylindrocarpon (DFRN03) and Epacris Juss., Caladenia R.Br., Cymbidium Sw., microphylla (isolates DFRN04 and Dendrobium Sw., Diuris Sm., Goodyera DFRN05), similar to Bayman and Otero R.Br., and Platanthera Rich. The other (2006) which listed a large amounts of

212 วารสารวิชาการเกษตร ปีที่ 33 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2558 fungal endophytes species isolated from months. The most advanced protocorm epiphytic, terrestrial and lithophytic development at stage 3 was promoted by orchids roots. isolate DFRN02 (Ceratobasidium sp.) for 4 months after seed sowing (Figure 3). The Symbiotic seed germination isolates DFRN04 (Cylindrocarpon) and In vitro symbiotic seed germination DFRN05 (Epacris microphylla) also is a powerful tool for both the production promoted seed germination at stage 1. of mycobiont-infected seedlings for use in Nevertheless, the difference between plant reintroduction and the study of percentages of symbiotic and asymbiotic fungal specificity within the Orchidaceae. seed germination was not high (15%), However, there are a few reports that and the final protocorm developmental address in vitro symbiotic seed germination stage is not sufficient for propagation and and development of Thai native orchid conservation of this epiphytic orchid species (Athipunyakom et al., 2004; species at present. There is a complicated Nonthachaiyapoom et al., 2011). This is relationship between orchid and endophytic the first study that describes in vitro fungi, and it is not clear whether the symbiotic seed germination of D. mycorrhizal fungi found in adult plants are formosum. Over 60% of seed in this the same fungi necessary for seed study germinated and developed to germination (Porras-Alfaro and Bayman protocorm after sowing for 3 months 2007). Moreover, some reports suggest (Table 3). After 4 months sowing, seed that there are specific endophytic fungi germination of DFRN02, DFRN04 and for different stages of growth in an DFRN05 treatment increased to 100%, orchid’s life cycle (Dearnaley, 2007). Also 89.13% and 83.89% respectively which it is possible that symbiosis may require were significantly higher than control. particular environmental conditions that Moreover, percentage of seed germination cannot be achieved in the laboratory to in treatments inoculated with fungal promote the symbiotic relationship isolates DFRN02 (100%), DFRN04 between orchid and fungus. Finding an (89.13%) and DFRN05 (83.89%) were effective mycobiont for D. formosum is a significantly different from control at 4 promising step forward in continuing

Thai Agricultural Research Journal Vol. 33 No. 2 May - August 2015 213 Table 1 Different seed germination stages of D. formosum. Stage Characteristics 0 No germination, seed with intact seed coat 1 Enlarged embryo, seed coat ruptured by the enlargement of embryo (=germination) 2 Continued embryo enlargement, rupture of testa 3 Appearance of protomeristem 4 Emergence of first leaf 5 Elongation of first leaf and further leaf development

Table 2 Morphology and molecular characteristics identification of five endophytic fungal isolated from the wild Dendrobium formosum orchid. Isolate Morphology Sequence match Identity (%) Fungal group Colony color on PDA was white to cream, aerial hyphae Cylindrocarpon sp. Hypocreales; DFRN01 97 in the center with white FJ605259 Nectriaceae submerged margin Colony color on PDA was creamy white, monilioid Ceratobasidium sp. Cantharellales; DFRN02 93 cells ellipsoidal, aerial and GU937735 Ceratobasidiaceae submerged hyphae Colony color on PDA was Cylindrocarpon sp. Hypocreales; DFRN03 white to cream, aerial and 98 FJ605259 Nectriaceae submerged hyphae Colony on PDA was white to Epacris microphylla Hypocreales; DFRN04 98 pale yellow with aerial hyphae AY268191 Cordycipitaceae Colony on PDA was white to cream with aerial hyphae, Epacris microphylla Hypocreales; DFRN05 dense, forming granular 97 aggregation in the center with AY268191 Cordycipitaceae submerged margin

214 วารสารวิชาการเกษตร ปีที่ 33 ฉบับที่ 2 พฤษภาคม - สิงหาคม 2558 Table 3 Percentages of seed germination and protocorm development of D. formosum seed after 1, 2, 3 and 4 months sowing Percentage of seed germination after Fungus 1 Months 2 Months 3 Months 4 Months Control 0.00 c 11.81 d 60.88 c 68.44 d DFRN01 2.56 c 3.63 d 70.44 c 71.63 cd DFRN02 15.00 a 77.31 a 100.00 a 100.00 a DFRN03 1.50 c 4.19 d 61.88 c 72.69 cd DFRN04 12.06 ab 60.69 b 84.13 b 89.13 ab DFRN05 6.94 a 42.44 c 70.31 c 83.89 bc In a column, mean followed by a common letter are not significantly different at 5% level by DMRT

efforts to develop reintroduction and growth in vitro. Orchid-fungal symbiosis conservation procedures for this rare and value in D. formosum propagation and their threatened orchid species, as the reintroduction into natural habitats. It can techniques employed here can be be applied to other endangered species. relevant to other endangered and Moreover, we now need to understand threatened wild orchid species. Further the efficacy of D. formosum symbiotic studies should focus on improving the seed germination insitu. If the orchid efficiency of D. formosum in situ seedling indeed requires different fungal strains development, such as mass propagation, within its lifetime, such knowledge about transplant success to nursery and then to the fungi partners will be helpful for the natural habitats. reintroduction and conservation of this species, as well as other commercial or CONCLUSION endangered and threatened Thai orchid species. An in situ baiting technique is Association between symbiotic quite promising to be further used in seed germination and mycorrhiza fungi of orchid conservation program. D. formosum was found. Only DFRN02 isolate, was a suitable mycobiont for D. ACKNOWLEDGEMENTS formosum seed germination and seedling This work was granted by

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