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Root exudates play an important role in rhizosphere interactions between plants and microorganisms. However, collection of chemicals from plant root system is difficult due to their low concentrations and high level of contaminants in growth media. The continuous collection method has been described in several terrestrial plants over the past 30 years (Tang and Young, 1982; Kong et al., 2004). Here, we describe a protocol for the collection of sterile root exudate from a floating aquatic plant, duckweed.

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Collection of Root Exudate from Duckweed
浮萍根部分泌物的采集

植物科学 > 植物新陈代谢 > 其它化合物
作者: Yufang Lu
Yufang LuAffiliation: Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Bio-protocol author page: a1894
Li Sun
Li SunAffiliation: Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Bio-protocol author page: a1895
 and Weiming Shi
Weiming ShiAffiliation: Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
For correspondence: wmshi@issas.ac.cn
Bio-protocol author page: a1896
Vol 5, Iss 1, 1/5/2015, 3753 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1367

[Abstract] Root exudates play an important role in rhizosphere interactions between plants and microorganisms. However, collection of chemicals from plant root system is difficult due to their low concentrations and high level of contaminants in growth media. The continuous collection method has been described in several terrestrial plants over the past 30 years (Tang and Young, 1982; Kong et al., 2004). Here, we describe a protocol for the collection of sterile root exudate from a floating aquatic plant, duckweed.
Keywords: Root exudate(根系分泌物), Duckweed(浮萍), Collection(收集), Plant-bacteria interactions(植物细菌的相互作用)

[Abstract]

Materials and Reagents

  1. Duckweed fronds, Spirodela polyrrhiza (collected from the paddy field drainage in the Tai Lake region of China)
  2. Sterile deionized water
  3. Methanol (HPLC grade) (Sigma-Aldrich, catalog number: 179337)
  4. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 653799)
  5. Formaldehyde solution (37 wt.% in H2O) (Sigma-Aldrich, catalog number: 252549)
  6. Sodium hypochlorite (NaClO) (available chlorine 4.00-4.99 %) (Sigma-Aldrich, catalog number: 239305)
  7. 0.15% formaldehyde solution (see Recipes)
  8. 0.5% v/v NaClO (see Recipes)
  9. Modified Steinberg nutrient solution (see Recipes)

Equipment

  1. Duckweed root exudate collection device (Figure 1)
    1. Plexiglass pot (D x H, 16 x 16 cm)
    2. Glass column (D x H, 23 x 170 mm)
    3. XAD-4 resin (20-60 mesh) (Sigma-Aldrich, catalog number: 37380-42-0)
    4. Hydrophobic fluoropore (PTFE) membrane (14 x 14 cm)
    5. Glass wool (Sigma-Aldrich, catalog number: 18421)
    6. Teflon stopper
    7. Aluminum foil
    8. Silicone tube


      Figure 1. Equipment used in root exudate collection

  2. Clean bench
  3. Peristaltic pump
  4. Rotary evaporator (Tokyo Rikakikai, EYELA, model: N-1100D)
  5. Freeze dryer (Freezone Plus 2.5) (Labconco)
  6. Incubation chamber (23 ± 1 °C, under fluorescent lamps at 100 μmol/m/s, 16 h light/8 h dark)

Procedure

  1. Preparation of the sterile duckweed fronds
    1. Wash 100 duckweed fronds with deionized water for 5 min. Transfer the fronds to a 500-ml flask with 200 ml of 0.5% v/v NaClO and stir gently for 30 min.
    2. Rinse the surface-sterilized plants five times with sterile deionized water. Transfer them to a 500-ml flask with 200 ml sterile modified Steinberg nutrient solution.
      Note: This should be performed in a clean bench.
    3. The fronds are statically grown in an incubation chamber at 23 ± 1 °C under fluorescent lamps at 100 μmol/m2/s (16 h light/8 h dark) until use (Figure 2).


      Figure 2. The incubation condition of the duckweed fronds

      Note: The duckweed fronds will gradually lose chlorophyll hours after sterilization (6 hours later). After a day, the growing points which are protected by the capsules remain green, whereas the other parts of the fronds become completely yellow. Three days later, new fronds are grown from the growing points, but smaller. They will recover completely after five days. Therefore, it will take about ten days for duckweed to grow normally after sterilization (Figure 3).


      Figure 3. The duckweed fronds at each stage after sterilization. A. Before sterilization. B. After 6 h. C. After 1 day. D. After 3 day. E. After 5 day. F. After 10 day.

  2. Preparation of the sterile root exudate collection device
    1. Soak the plexiglass pot, glass column and silicone tube in the 0.15% formalin solution overnight. Wash them with sterile deionized water.
    2. Soak the glass wool in the 2 M HCl overnight, wash it using sterile deionized water three times to pH 7.0, then autoclave it.
    3. Wash the XAD-4 resin with sterile deionized water. Elute the resin with methanol. Store the clean resin in methanol in a dark glass container until use.
    4. The glass column is fixed on an iron support. Put about 0.2 g of glass wool at the bottom to prevent resin from leaking with a glass rod (Figure 4A). Connect a 5-cm long silicone tube to the bottom of glass column (Figure 4B).
    5. Stir the XAD-4 resin and pack the column with 60 ml soaked resin (Figure 4C-D). Remove the residual methanol by washing the column with about 1.5 L of sterile deionized water.
      Note: Keep the column vertical to avoid bias flow. Pour the resin into column slowly to avoid bubbles.
    6. Fasten the tube with a clip to avoid resin column becoming dry (Figure 4E).
    7. Keep the water and resin level tangent (Figure 4F). Put about 0.2 g of glass wool on the top of resin to isolate from the air and fix the resin (Figure 4G).
    8. Connect the column to the top of the pot through a perforated Teflon stopper. Wrap the column with aluminum foil to restrict light and deter algae growth (Figure 4H).
    9. Connect a peristaltic pump to the bottom of the pot with a silicon tube.


      Figure 4. Preparing and packing the column. A, B. Put some glass wool at the bottom and connect with a silicone tube. C, D. Stir the XAD-4 resin and pack the column. E. Fasten the tube with a clip. F, G. Keep the water and resin level tangent and put some glass wool on the top. H. Wrap with aluminum foil.

  3. Collection of duckweed root exudate
    1. Wash 100 cm2 (about 2 g fresh weight, 50% coverage) of the sterile duckweed fronds twice with sterile water. Transplant them into the pot containing 2 L sterile nutrient solution. Cover the pot with an autoclaved hydrophobic fluoropore (PTFE) membrane to maintain a sterile environment.
      Note: This should be performed on a clean bench.
    2. Begin the chemical trapping. The nutrient solution circulates at a rate of 10 ml/min by the peristaltic pump. The exudates are continuously attached to the resin.
    3. After five days, detach the column with 1.5 L of deionized water. Elute it with 150 ml methanol.
    4. Evaporate the methanol under vacuum at 40 °C. Freeze-dry the remaining aqueous solution, re-dissolve the residue in 1 ml methanol, store at -20 °C.


      Figure 5. The continuous duckweed root exudate-trapping system (Lu et al., 2014). A. A real picture of the collection system. B. A diagram of the collection system. The nutrient solution was continuously circulated through the root systems of the duckweed plants, eluting the duckweed-derived organic compounds. Hydrophobic or partially hydrophobic exudates were selectively absorbed by the XAD-4 resin while the inorganic nutrients were recycled to sustain duckweed growth.

Recipes

  1. 0.15% formaldehyde solution
    Mix 20 ml of formaldehyde solution in 5 L deionized water
  2. 0.5% v/v NaClO
    Mix 1 ml of NaClO in 199 ml deionized water
    Stored at 4 °C
  3. Modified Steinberg nutrient solution
    0.42 mM Ca (NO3)2.4H2O
    0.23 mM NH4Cl
    0.41 mM MgSO4.7H2O
    12.9 μM KH2PO4
    0.63 μM ZnSO4.7H2O
    0.91 μM MnCl2.4H2O
    1.94 μM H3BO3
    0.18 μM NaMoO4.2H2O
    2.80 μM FeCl3.6H2O
    2.80 μM Na2EDTA.2H2O
    Adjusted to pH 6.8 by 1 M NaOH
    Autoclave
    Stored at room temperature

Acknowledgments

This work was supported by grants from the Strategic Priority Research Program (B)-“Soil-Microbial System Function and Regulation” of the Chinese Academy of Sciences (XDB15030100) and the National Science Technology Program (2012BAD15B03). This protocol was published in our Nature paper (Lu et al., 2014).

Reference

  1. Kong, C., Liang, W., Xu, X., Hu, F., Wang, P. and Jiang, Y. (2004). Release and activity of allelochemicals from allelopathic rice seedlings. J Agric Food Chem 52(10): 2861-2865.
  2. Lu, Y., Zhou, Y., Nakai, S., Hosomi, M., Zhang, H., Kronzucker, H. J. and Shi, W. (2014). Stimulation of nitrogen removal in the rhizosphere of aquatic duckweed by root exudate components. Planta 239(3): 591-603.
  3. Tang, C. S. and Young, C. C. (1982). Collection and Identification of Allelopathic Compounds from the Undisturbed Root System of Bigalta Limpograss (Hemarthria altissima). Plant Physiol 69(1): 155-160.

材料和试剂

  1. 浮萍叶, (从中国太湖地区的稻田排水渠收集)
  2. 无菌去离子水
  3. 甲醇(HPLC级)(Sigma-Aldrich,目录号:179337)
  4. 盐酸(HCl)(Sigma-Aldrich,目录号:653799)
  5. 甲醛溶液(37重量%的H 2 O)(Sigma-Aldrich,目录号:252549)
  6. 次氯酸钠(NaClO)(有效氯4.00-4.99%)(Sigma-Aldrich,目录号:239305)
  7. 0.15%甲醛溶液(见配方)
  8. 0.5%v/v NaClO(参见配方)
  9. 改良的Steinberg营养液(见配方)

设备

  1. 浮萍根分泌物收集装置(图1)
    1. 有机玻璃罐(D×H,16×16cm)
    2. 玻璃柱(D×H,23×170mm)
    3. XAD-4树脂(20-60目)(Sigma-Aldrich,目录号:37380-42-0)
    4. 疏水性氟孔(PTFE)膜(14×14cm)
    5. 玻璃棉(Sigma-Aldrich,目录号:18421)
    6. 特氟龙塞子
    7. 铝箔
    8. 硅胶管


      图1.在根系分泌物收集中使用的设备

  2. 清洁长椅
  3. 蠕动泵
  4. 旋转蒸发器(Tokyo Rikakikai,EYELA,型号:N-1100D)
  5. 冷冻干燥机(Freezone Plus 2.5)(Labconco)
  6. 孵育室(23±1℃,荧光灯下100μmol/m/s,16小时光照/8小时黑暗)

程序

  1. 无菌浮萍叶片的制备
    1. 用去离子水洗涤100棵浮萍叶片5分钟。 转移 叶片置于具有200ml的0.5%v/v NaClO的500-ml烧瓶中并轻轻搅拌 30分钟。
    2. 冲洗表面消毒的植物五次 无菌去离子水。 将其转移到具有200ml的500ml烧瓶中 无菌改良Steinberg营养液 注意:这应该在一个干净的工作台上进行。
    3. 叶片在23±1℃的孵育室中静止生长 在荧光灯下以100μmol/m 2/s/s(16小时光照/8小时黑暗)直至 使用(图2)。


      图2.浮萍叶状体的孵育条件

      注意:浮萍叶片会在几小时后逐渐失去叶绿素 灭菌(6小时后)。 一天后,生长点是   保护胶囊保持绿色,而其他部分 叶片变成完全黄色。 三天后,新的叶状体生长 从增长点,但更小。 他们将完全恢复   五天。 因此,浮萍将需要大约十天的时间 通常在灭菌后(图3)。


      图3.浮萍 灭菌后每个阶段的叶状体。 A。 灭菌前。 乙。 6小时后。 C. 1天后。 D.3天后。 E.5天后。 F.10之后 天。

  2. 无菌根分泌物收集装置的制备
    1. 将有机玻璃罐,玻璃柱和硅胶管浸泡在0.15% 福尔马林溶液过夜。 用无菌去离子水洗涤它们
    2. 将玻璃棉浸泡在2M HCl中过夜,用无菌洗涤 去离子水3次,至pH 7.0,然后高压灭菌。
    3. 洗 XAD-4树脂用无菌去离子水。 用树脂洗脱树脂 甲醇。 将清洁的树脂储存在黑色玻璃容器中的甲醇中 直到使用。
    4. 玻璃柱固定在铁支架上。 放 在底部约0.2g玻璃棉,以防止树脂泄漏 与玻璃棒(图4A)。 将5厘米长的硅胶管连接到 玻璃柱底部(图4B)。
    5. 搅拌XAD-4树脂和包装   该柱用60ml浸泡的树脂(图4C-D)。 删除残留物 甲醇通过用约1.5L无菌去离子洗涤柱 水。
      注意:保持列垂直,以避免偏差流。 缓慢倒入树脂,避免气泡。
    6. 用夹子固定管子,以避免树脂柱变干(图4E)。
    7. 保持水和树脂水平切线(图4F)。 放0.2克 的玻璃纤维顶部的树脂隔离空气和固定 树脂(图4G)。
    8. 将色谱柱连接到锅的顶部 通过穿孔的特氟隆塞。 用铝箔包裹柱子 以限制光和阻止藻类生长(图4H)
    9. 用硅管将蠕动泵连接到锅底。


      图4.准备和包装色谱柱。 A,B.放一些玻璃棉 在底部并与硅胶管连接。 C,D搅拌XAD-4 树脂并包装柱。 E.用夹子固定管子。 F,G. Keep 水和树脂水平切线,并在上面放一些玻璃棉。 H。   包装用铝箔。

  3. 收集浮萍根渗出物
    1. 洗涤100cm 2(约2g鲜重,50%覆盖)的无菌 浮萍叶片两次用无菌水。 移植他们到锅里 含2 L无菌营养液。 用一个盖子锅 高压灭菌疏水性荧光(PTFE)膜保持无菌 环境 注意:这应该在一个干净的工作台上进行。
    2. 开始化学捕集。 营养液以一定速率循环   为10毫升/分钟的蠕动泵。 渗出物是连续的 附着在树脂上
    3. 五天后,用1.5L去离子水分离柱子。 用150ml甲醇洗脱。
    4. 在40℃下真空蒸发甲醇。 冷冻干燥 剩余水溶液,将残余物重新溶解在1ml甲醇中, 储存于-20°C。


      图5.连续浮萍根 渗出物捕获系统(Lu et al。,,2014)。 收集系统。 B.收集系统图。 营养 溶液连续循环通过根系 浮萍植物,洗脱浮萍衍生的有机化合物。 疏水性或部分疏水性渗出物被选择性吸收 通过XAD-4树脂,而无机营养物被回收 维持浮萍生长。

食谱

  1. 0.15%甲醛溶液
    混合20ml甲醛溶液在5L去离子水中
  2. 0.5%v/v NaClO/v 将1ml NaClO溶于199ml去离子水中
    储存在4°C
  3. 改良的Steinberg营养液
    0.42mM Ca(NO 3)2 Sub 2 O 4/4H 2 O 2 / 0.23mM NH 4 Cl/s 0.41mM MgSO 4。7H 2 O 2 12.9μMKH sub 2 PO 4 sub
    0.63μMZnSO 4 sub 7H O
    0.91μMMnCl 2 4H 2 O 1.94μMH sub 3 BO 3
    0.18μMNaMoO 4 2H O 2.80μMFeCl 3 3 6H 2 O O 2.80μMNa 2 EDTA EDTA。 2H 2 O 用1M NaOH调节至pH 6.8 高压灭菌器
    在室温下贮存

致谢

这项工作得到了中国科学院"土壤微生物系统功能与调节"(XDB15030100)和国家科学技术计划(2012BAD15B03)战略重点研究计划(B)的资助。 这个协议发表在我们的自然论文(Lu 等人,2014)。

参考

  1. Kong,C.,Liang,W.,Xu,X.,Hu,F.,Wang,P.and Jiang,Y。 从化感水稻幼苗释放和活性化合物。 Agric Food Chem 52(10):2861-2865。
  2. Lu,Y.,Zhou,Y.,Nakai,S.,Hosomi,M.,Zhang,H.,Kronzucker,H.J。和Shi,W。 根系分泌物成分刺激水生浮萍根际氮的除氮。 Planta 239(3):591-603。
  3. Tang,C.S。和Young,C.C。(1982)。 从Bigalta Limpograss的不受干扰的根系统收集和鉴定对映异构化合物( Hemarthria altissima 69 69(1):155-160。
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How to cite this protocol: Lu, Y., Sun, L. and Shi, W. (2015). Collection of Root Exudate from Duckweed. Bio-protocol 5(1): e1367. DOI: 10.21769/BioProtoc.1367; Full Text



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