Establishment of a Symbiotic in vitro System between a Green Meadow Orchid and a Rhizoctonia-like Fungus

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Jun 2014



Symbiotic orchid seed germination in an in vitro system allows the growth of mycorrhizal protocorms and plantlets for scientific purposes. Orchids in nature need to establish a mycorrhizal symbiosis with fungal partners to germinate and develop into adult plants. Here we present a protocol for symbiotic germination of the terrestrial Mediterranean green meadow orchid Serapias vomeracea. The fungal symbiont Tulasnella calospora (T. calospora) (Basidiomycetes, Cantharellales) was chosen because of its common occurrence (Girlanda et al., 2011), its ability to grow in culture and compatibility in germination assays. T. calospora is one of the most common rhizoctonia-like fungi associated with terrestrial as well as epiphytic orchids.

Materials and Reagents

  1. Serapias vomeracea seeds (collected in the field, in the north-west Mediterranean meadows of Italy; seeds from this orchid species could be retrieved through “index seminum” of international botanic gardens or germplasm banks)
  2. Tulasnella calospora inoculum (strain MUT 4182) (previously grown in sterile culture on MEA medium)
  3. Sterile double deionized water (neutral pH)
  4. Tween-20 (Sigma-Aldrich, catalog number: P2287 )
  5. Milled oats (retrieved from organic shops)
  6. Malt Extract (Sigma-Aldrich, Fluka, catalog number: 70167 )
  7. Ethanol (Sigma-Aldrich, Fluka, catalog number: 0 2860 )
  8. 5% sodium hypochlorite (commercial bleach)
  9. Agar (Sigma-Aldrich, catalog number: A1296 )
  10. Glucose (Sigma-Aldrich, catalog number: G8270 )
  11. Peptone (Sigma-Aldrich, Fluka, catalog number: P5905 )
  12. Sterilizing solution (see Recipes)
  13. Solid oat medium (see Recipes)
  14. MEA medium (see Recipes)


  1. Balance
  2. Small spatula
  3. Small metal forceps
  4. Sterile 10 x 10 mm Whatman No.1 filter paper
  5. Lancets
  6. 2.0 ml centrifuge tubes
  7. Micropipette
  8. Filter tips
  9. Centrifuge
  10. Vortex
  11. Timer
  12. Laminar flow hood
  13. Petri dishes (9 cm diameter)
  14. Parafilm (Sigma-Aldrich, catalog number: P7793 )
  15. Bunsen burner
  16. Climatic chamber (Binder GmbH)


  1. Wipe the working area with ethanol and alcohol/flame sterilize the spatula.
  2. Wipe the laminar flow hood and alcohol/flame sterilize the forceps and the lancets. All steps must be carried out under laminar flow hood.
  3. Fill about 25 mg of Serapias vomeracea seeds for each 2.0 ml centrifuge tube, being careful to bring down all the seeds to the bottom of the centrifuge tube. For this purpose, centrifuge all the tubes at maximum speed with their lids open for 1 min. Make sure not to leave any remains of seeds on the cap edge; these seeds will not be sterilized and may contaminate those sterilized.
  4. Sterilize seeds adding 1 ml of sterilizing solution for each tube and start immediately the timer; mix well and place on vortex for 18 min.
  5. After this, centrifuge at maximum speed for 1 min and, quickly replace Sterilizing solution with 1 ml of sterile deionized water. Incubate for 5 min and repeat this washing step three times. To avoid collecting seeds in the tip while change washing water, briefly centrifuge to bring down all the seeds to the bottom of the centrifuge tube.
    Note: Steps 4 and 5 must be completed within 20 min.
  6. Using sterile forceps, spread 4-10 squares of Whatman No.1 filter paper (10 x 10 mm) spaced equally on a petri dish filled with 25 ml of Solid oat medium as shown in the Figure 1. Gently place the seeds spread out on the filter paper squares using pipette and filter tips. Normally it is recommended to prepare from 10 to 20 petri dishes.
    Note: each filter piece of paper will be a replicate and the number of squares indicated above is the minimum and the maximum to use in a single petri dish, as how many replicates you need.
  7. Inoculate each petri dish with Tulasnella calospora (AL13/D strain). Inoculum consist of a 3 x 3 mm portion of actively growing mycelium (from pure culture on MEA medium) cut with sterile lancets from the petri dish of the fungal pure culture, placed in the center of capsule using sterile lancets (Figure 1). Control plates were left uninoculated.
  8. Seal petri dishes with Parafilm and aluminum sheet (to ensure darkness) and place in a climatic chamber at stable temperature of 20 °C (Figure 2).
  9. Seeds are considerable to be germinated when protocorms developed from imbibed seeds after 15-30 days. After this time, well developed protocorms, start to grow in plantlets with leaves. Well-developed plantlets may be exposed at light with photoperiod of 16 h of light (20-25 °C) and 8 h of dark (18-20 °C) (Figure 3).

Representative data

Figure 1. Representative scheme of germination assay [modified from Ercole (2014)]

Figure 2. Developed protocorms after 15-30 days of dark incubation

Figure 3. Developed protocorms after 45 days (30 days of darkness and 15 days of light exposure)

The reproducibility of the symbiotic seed germination of S. vomeracea with T. calospora is usually successful. In some cases we noted low germination percentage due to seed viability mainly. Viability test on seeds allow to evaluating a priori the success rate. Percentage of germination can be evaluated by count of all seeds at time zero (all seeds sown) and the germinated seeds at time 15, 30 and 45 days. Usually we reported high percentage of germination (from 95% at time 15 days, to 60% at time 45 days). The decrease of the percentage of germination from time 15 days to time 45 days is mainly due to “natural selection” and/or competition between seeds (seeds with a high growth rate grow at the detriment of slower).


Note that this protocol can be applied to many different orchid and fungus species, with some modification in the sterilization time of seeds. Every orchid species has different seeds with different teguments, so the sterilization time depends on this. In some cases success of germination depends on compatibility between orchid and fungus; indeed, not all fungi (also in the Rhizoctonia-form genus) can germinate orchid seeds.


  1. Sterilizing solution (1% sodium hypochlorite) (100 ml)
    Sodium hypochlorite 5% (w/v) 20 ml
    Add deionized water to 100 ml final volume
    Stir well to mix
    Add 100 µl of Tween-20 to the solution
  2. Solid oat medium (0.3% milled oats, 1% agar) (1 L)
    Milled oats 3 g
    Agar 10 g
    Add deionized water to 1 L final volume
    Autoclave for 20 min
  3. MEA medium (1.8% malt extract, 2.0% agar, 2.0% glucose, 0.2% peptone) (1 L)
    Malt Extract 18 g
    Agar 20 g
    Glucose 20 g
    Peptone 2 g
    Add deionized water to 1 L final volume
    Autoclave for 20 min


This protocol was adapted from the following publications: Perotto et al. (2014); Ercole et al. (2013); Ercole (2014).
Research on orchid mycorrhiza was partly funded by the Italian MIUR and by local funding from the University of Torino.


  1. Ercole, E. (2014). Micorrize e conservazione delle orchidee. In: Adamo, M., Masella, E. A., Bonini, I., Borzatti, A., Buono, S., Carasso, V., Castagnini, P., Ceriani, R. M., Chioccia, G., Ciaschetti, G., Vitis, M. D., Martino, D. L., Ercole, E., Fabrini, G., Fay, M. F., Fonck, M., Gallino, B., Gransinigh, E., Haile, G., Legitimo, I., Maccherini, S., Magrini, S., Mannocci, M., Marks, T. R., Mazzoncini, V., Pellegrino, G., Pierce, S. and Pirondini, A. (eds). Esperienze di conservazione delle orchidee. Viterbo: Tipoligrafia Quatrini, vol. 1, p. 17-23.
  2. Ercole, E., Rodda, M., Molinatti, M., Voyron, S., Perotto, S. and Girlanda, M. (2013). Cryopreservation of orchid mycorrhizal fungi: a tool for the conservation of endangered species. J Microbiol Methods 93(2): 134-137.
  3. Girlanda, M., Segreto, R., Cafasso, D., Liebel, H. T., Rodda, M., Ercole, E., Cozzolino, S., Gebauer, G. and Perotto, S. (2011). Photosynthetic Mediterranean meadow orchids feature partial mycoheterotrophy and specific mycorrhizal associations. Am J Bot 98(7): 1148-1163.
  4. Perotto, S., Rodda, M., Benetti, A., Sillo, F., Ercole, E., Rodda, M., Girlanda, M., Murat, C. and Balestrini, R. (2014). Gene expression in mycorrhizal orchid protocorms suggests a friendly plant-fungus relationship. Planta 239(6): 1337-1349.


在体外系统中的共生兰花种子萌发允许用于科学目的的菌根原球茎和小植物的生长。 兰花本质上需要建立与真菌合作者的菌根共生,以发芽和发育成成虫植物。 在这里我们提出了地中海地中海绿色草地兰花 Serapias vomeracea的共生萌发的协议。 选择真菌共生菌(Talsnella calospora)( T。calospora )(担子菌门,Cantharellales),因为其常见的发生(Girlanda等人,2011 ),其在培养中生长的能力和在发芽测定中的相容性。 T。 calospora 是与陆生以及附生兰花相关的最常见的丝孢菌样真菌之一。


  1. 种子(在田间收集,在意大利的西北地中海草甸;来自该兰花种子的种子可以通过国际植物园的索引seminum 检索 或种质库)
  2. 接种物(菌株MUT 4182)(预先在MEA培养基上的无菌培养物中生长)
  3. 无菌双去离子水(中性pH)
  4. 吐温-20(Sigma-Aldrich,目录号:P2287)
  5. 碾碎的燕麦(从有机商店检索)
  6. 麦芽提取物(Sigma-Aldrich,Fluka,目录号:70167)
  7. 乙醇(Sigma-Aldrich,Fluka,目录号:02860)
  8. 5%次氯酸钠(商业漂白剂)
  9. 琼脂(Sigma-Aldrich,目录号:A1296)
  10. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  11. 蛋白胨(Sigma-Aldrich,Fluka,目录号:P5905)
  12. 灭菌溶液(参见配方)
  13. 固体燕麦培养基(见配方)
  14. MEA介质(参见配方)


  1. 余额
  2. 小铲
  3. 小金属钳
  4. 无菌10 x 10毫米Whatman 1号滤纸
  5. 柳叶刀
  6. 2.0 ml离心管
  7. 微量移液器
  8. 过滤提示
  9. 离心机
  10. 涡流
  11. 计时器
  12. 层流罩
  13. 培养皿(直径9cm)
  14. 石蜡膜(Sigma-Aldrich,目录号:P7793)
  15. 本生灶
  16. 气候室(Binder GmbH)


  1. 用乙醇和酒精擦拭工作区域,用火焰对抹刀消毒
  2. 擦拭层流罩和酒精/火焰消毒镊子和柳叶刀。 所有步骤必须在层流罩下进行
  3. 每个2.0ml离心管中加入约25mg的种子,仔细地将所有的种子放到离心管的底部。 为此,将所有管以最大速度离心,其盖子打开1分钟。 确保不要在盖边缘留下任何种子的残留; 这些种子将不会被灭菌并且可能污染那些灭菌的那些。
  4. 消毒种子,每个管加入1ml灭菌溶液,立即启动计时器;混匀,并置于涡旋18分钟。
  5. 之后,以最大速度离心1分钟,并用1ml无菌去离子水快速更换灭菌溶液。孵育5分钟,并重复该洗涤步骤三次。为了避免在更换洗涤水时在尖端中收集种子,简要离心以将所有种子收集到离心管的底部。
  6. 使用无菌镊子,在填充有25ml如图1所示的固体燕麦培养基的培养皿上均匀分布4-00平方的Whatman No.1滤纸(10×10mm)。轻轻地将种子铺开滤纸方格使用吸管和过滤嘴。通常建议准备10至20个培养皿。
  7. 接种每个陪替氏培养皿用 Calsalella calospora (AL13/D菌株)。接种物由用来自真菌纯培养物的培养皿的无菌柳叶刀切割的活性生长的菌丝体(来自MEA培养基上的纯培养物)的3×3mm部分,使用无菌柳叶刀置于胶囊的中心(图1)。对照板未接种。
  8. 密封培养皿与石蜡膜和铝板(以确保黑暗),并放置在温度稳定在20℃的气候室(图2)。
  9. 当在15-30天后从吸收的种子形成原球茎时,种子相当可能萌发。在这段时间后,发达的原球茎,开始生长在带叶子的小植物。良好发育的苗可以暴露在光周期为16小时光照(20-25°C)和8小时黑暗(18-20°C)(图3)的光照下。




图3. 45天(30天黑暗和15天曝光)后开发的原生动物

共生种子萌发的重复性。 vomeracea 与 T。 calospora 通常是成功的。在一些情况下,我们注意到由于种子活力主要的低发芽率。对种子的活力测试允许先验地评估成功率。发芽百分比可以通过在时间零点(所有种子播种)的所有种子和在15,30和45天的发芽种子的计数来评估。通常我们报告高发芽率(从15天时的95%到45天时的60%)。从时间15天到时间45天的萌发百分比的减少主要是由于"自然选择"和/或种子之间的竞争(具有高生长速率的种子以较慢的速度生长)。




  1. 灭菌溶液(1%次氯酸钠)(100ml) 次氯酸钠5%(w/v)20ml
    加入去离子水至100 ml终体积 搅拌均匀混合
  2. 固体燕麦培养基(0.3%研磨燕麦,1%琼脂)(1L)
    加入去离子水至1 L最终体积
  3. MEA培养基(1.8%麦芽提取物,2.0%琼脂,2.0%葡萄糖,0.2%蛋白胨)(1L)
    蛋白胨2 g
    加入去离子水至1 L最终体积


该协议改编自以下出版物:Perotto等人(2014); Ercole (2013); Ercole(2014)。


  1. Ercole,E。(2014)。 Micorrize e conservazione delle orchidee。 In:Adamo,M.,Masella,EA,Bonini,I.,Borzatti,A.,Buono,S.,Carasso,V.,Castagnini,P.,Ceriani,RM,Chioccia,G.,Ciaschetti, Vitis,MD,Martino,DL,Ercole,E.,Fabrini,G.,Fay,MF,Fonck,M.,Gallino,B.,Gransinigh,E.,Haile,G.,Legitimo,I.,Maccherini,S 。,Magrini,S.,Mannocci,M.,Marks,TR,Mazzoncini,V.,Pellegrino,G.,Pierce,S.and Pirondini,A。 Esperienze di conservazione delle orchidee 。 Viterbo:Tipoligrafia Quatrini,vol。 1,p。 17-23。
  2. Ercole,E.,Rodda,M.,Molinatti,M.,Voyron,S.,Perotto,S.and Girlanda,M。(2013)。 冷冻保存兰花菌根真菌:保护濒危物种的工具 J Microbiol Methods 93(2):134-137。
  3. Girlanda,M.,Segreto,R.,Cafasso,D.,Liebel,H.T.,Rodda,M.,Ercole,E.,Cozzolino,S.,Gebauer,G.and Perotto, 光合地中海草甸兰花具有部分肌营养不良和特异性菌根关系。 Bot 98(7):1148-1163。
  4. Perotto,S.,Rodda,M.,Benetti,A.,Sillo,F.,Ercole,E.,Rodda,M.,Girlanda,M.,Murat,C.and Balestrini,R。 在菌根兰花原生动物中的基因表达提示了一种友好的植物真菌关系。 Planta 239(6):1337-1349。
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引用:Ercole, E., Rodda, M., Girlanda, M. and Perotto, S. (2015). Establishment of a Symbiotic in vitro System between a Green Meadow Orchid and a Rhizoctonia-like Fungus. Bio-protocol 5(10): e1482. DOI: 10.21769/BioProtoc.1482.