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[Bio101] Arabidopsis Growing Protocol – A General Guide
[Bio101] 拟南芥培育方法—通用指南

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Abstract

Arabidopsis as the model organism for higher plants is widely studied among plant biology labs around the world. However, taking care of this tiny plant may not be trivial. Here is a general guide used for the Heven Sze lab at the University of Maryland, College Park. A lot of efforts have been taken by the Sze lab and fellow lab members to formulate a general guide for Arabidopsis plant growth in the lab.

Materials and Reagents

  1. Arabidopsis seed
  2. Miracle-Gro® Potting Mix with Fertilizer (2 cubic feet bag) (The Scotts Miracle-Gro Company, model: 74278300 from Lowe’s, Item: 156581)

  3. Miracle-Gro® 8 Qt. Miracle Gro® Perlite (The Scotts Miracle-Gro Company, model: 70752300 , Lowe’s, Item: 68468)
  4. 0.1% Triton X-100
  5. Deionized water (D.I. water)
  6. Sterile D.I. water
  7. 70% ethanol
  8. Drierite
  9. KNO3
  10. FeNa-EDTA
  11. KH2PO4
  12. MgSO4
  13. Ca(NO3)2.4H2O
  14. Sterilization solution (see Recipes)
  15. 1/4 Strength Hoagland’s solution (see Recipes)
  16. Homemade MS (Murashige and Skoog, 1962) medium for Arabidopsis plants under different Ca2+/Mn2+ conditions  (see Recipes)

Equipment

  1. The Scotts Miracle-Gro Company
  2. Autoclave bin
  3. Scooper
  4. Fisher dishes
  5. Pots (14 x 8.5 x 6 cm)
  6. Aluminum foil
  7. Humidome
  8. Sealed container
  9. Nylon mesh
  10. Autoclave tape
  11. Growth chamber
  12. Microcentrifuge
  13. Small Fisher dishes (100 x 15 mm size)
  14. Whatman filter paper

Procedure

  1. Preparing soil for planting soil
    We use Miracle-Gro® Potting Mix with Fertilizer and Miracle-Gro® 8 Qt. Miracle Gro® Perlite. To make Arabidopsis-friendly soil, mix well 1 bag of potting mix (2 cb ft) with half bag of perlite (4 qt) in a big bin used to store soil.
    Note: We always sterilize the soil before putting it into planting pots (see below).
    1. Scoop dry soil into an autoclave bin. Fill until the soil is approximately one inch from the top after packing down with the scooper.
    2. Cover the bins with aluminum foil and put a piece of autoclave tape on each one. Make sure the outsides of the bins are dirt free before putting in the autoclave.
    3. Autoclave on normal/flash/quick mode for 30-50 min depending on how many bins are being autoclaved.
    4. Once cool, the soil can be placed into pots, packing down lightly. Remember to cover the autoclaved soil well again to prevent any seed/fungi contamination. Once opened, autoclaved soil can be good for use for up to 2 weeks. Please don’t autoclave more than needed.
    5. Fill the flat with pots (not on the top of soil) with deionized water, cover with humidomes and let soak for 3-6 h or overnight. There should be standing water (~ 1 inch) after soaking, add more water if not. 
    6. The soil in pots is now ready for growing plants.

  2. Seeds sterilization
    1. Method A
      1. Put certain amount of seeds (wild type, mutant, or complements lines) into an eppendorf tube. Add 1 ml sterilization solution.
      2. Vortex at maximal speed for 20-30 sec.
      3. Stand for 7-10 min with occasional vortex.
      4. Spin in a microcentrifuge at 8,000 rpm for 5 sec.
      5. Pour off the water carefully.
      6. Add 1 ml sterile water and vortex to suspend the fluffy seeds.
      7. Spin in a microcentrifuge at 8,000 rpm for 5 sec and pour off the supernatant.
      8. Repeat steps 6-7 for another 4 times.
      9. Move the sterile seeds to small Fisher dishes with a wet Whatman filter paper (sterile or autoclaved) on its bottom.
      10. Transfer the seeds to agar medium plate or to soil with a tweezer that has been soaked in 70% ethanol for >30 min.
    2. Method B
      1. Put certain amount of seeds (no more than 0) into 1.5 ml Eppendorf tubes. Add 1 ml 70% ethanol.
      2. Pour off water and add 5 ml 70% ethanol, incubate for 5 min.
      3. Rinse with sterile D.I. water 4 times.
      4. Add 0.1% Triton X-100 (or 30% Bleach + 0.1% Triton X-100) 10 min.
      5. Rinse thoroughly with sterile D.I. water (at least 5 times).
      6. Move the sterile seeds to small Fisher dishes with a wet Whatman filter paper on its bottom.

  3. Planting
    1. General concern
      Please keep in mind your seeds can be contaminated with fungi and bacteria when they were produced on parental plants, seed sterilization is recommended for all plants (up to bolting time) and necessary for all flower-producing/seed-collecting plants and seeds from other labs. Chamber contamination is usually from dirty seeds in the lab (since we always use autoclaved soil). Flower parts are very attractive to insects and pests, please keep an eye on the flowers.
    2. Grow healthy Arabidopsis plants
      Before start, the trays must be cleaned/brushed free of old seeds or fungus.
      1. Fill pots (14 x 8.5 x 6 cm) to the top with autoclaved soil.
      2. Add D.I. water to the tray which contains the pots, about 1/3-1/2 the height of a pot. It usually takes at least 3 h to let the soil soak thoroughly. Then spray the surface of the soil with a spray bottle (D.I. water), to make sure that the seeds will fall on a wet surface.
      3. Transfer seeds to soil (8-10 seeds/pot, to space seeds apart).
      4. After planting, cover the tray with a humidome, taping it to the tray, to keep the necessary humidity.
      5. Put the tray in 4 °C cold room for 3-5 days (5 day is better.) at dark.
      6. Move to growth chamber with the humidome still covering.
      7. Plants should germinate after 3-4 days in chamber with long day light cycle. About 5-7 days leave the humidome half-open for anther 2-3 days when 2 pieces of cotyledons have developed completely.
      8. Remove the humidome and water with D.I. water twice a week (Tuesday and Friday, for example).

Notes

  1. Arabidopsis is sensitive to all stress conditions, so please water only when there is no standing water in the tray.
  2. If the plants are desired to proceed to flowering, water with 1/4 strength of Hoagland solution once every other week until they start bolting (twice in total is enough). Hoagland is nitrogen-rich media that promotes the transition to reproductive growth.
  3. Miracle-Gro potting soil has Micracle-Gro plant food (slowly-released fertilizer), which usually support plant growth for up to 3 months. So restrain the impulse to over-fertilize them.
  4. Keep an eye when plants start flowering. Use any possible pest control as needed.
  5. Aphids is a common insect problem. Spray 1:100 “Orthene” on the infected plants under hood, repeat spray for 3 days. Be careful not to over-spray them, otherwise it will kill the plants.
  6. When plants stop flowering, move them out to a ventilated area for seeds to dry. Collect seeds promptly as soon as the siliques look yellow or brown, break with little or no applied pressure.
  7. First, you should clean up some area free seeds of other species, to make sure all the seeds here are no contamination with other seeds you do not want. Rub your fingers around the silique, allow the seeds to fall on a big piece of paper below. Filter seeds through nylon mesh into another piece of paper. Store seeds in Eppendorf tubes, poke a hole on the cap of the tube. The collected seeds should be left at room temperature for one more week to dry. Then place them in a sealed container with Drierite.

Recipes

  1. Sterilization solution
    20% Chlorox bleach
    0.05% Tween 20
    In sterile water, must be freshly made.
  2. 1/4 Strength Hoagland’s solution (used to water Arabidopsis plant)

    FW
    Stock
    g/250 ml stock
    final
    ml stock/4 L final
    KNO3
    101.11
    2 M
    50.6
    1.25 mM
    2.5
    FeNa-EDTA
    367.1 20 mM
    1.8355
    12.5 μM
    KH2PO4
    136.1
    1 M
    34.0
    0.5 mM
    2.0
    MgSO4
    120.4
    2 M
    60.2
    0.5 mM
    1.0
    Ca(NO3)2.4H2O*
    236.2
    2 M
    118.1
    0.5 mM
    1.0
    Minors




    1.0
    Note: *Add last when volume is almost full.
    Minors
    Chemical
    FW
    Stock (mM)
    Final (μM)
    g/ 100 ml stock
    H3BO3
    61.83
    70
    17.5
    MnCl2
    197.9
    14
    3.5
    0.28  g
    CuSO4
    159.6
    0.5
    0.125
    0.08 g
    ZnSO4
    287.5
    1
    0.25
    0.03 g
    Na2MoO4•2H2O
    241.9
    0.2
    0.05
    0.005 g


    Note:  Start from >3.9 L water, then add every thing and stir thoroughly.
    Ca2+ may precipitate with SO42-.
  3. Homemade MS medium for Arabidopsis plants under different Ca2+/Mn2+ conditions
    Stock:
    a.  Macronutrients (10x)
    mM (1x MS)
    Salt
    10x stock (g/L)
    41.2
    NH4NO3
    16.5
    18.8
    KNO3
    19
    3.0
    CaCl2•anhydrous*
    3.3
    1.5
    MgSO4•7H2O
    3.7
    1.25
    KH2PO4
    1.7
    *CaCl2 is dropped out for Ca related medium.
    b.  Fe-EDTA (100x)
    mM (1x MS)
    Salt
    100x stock (g/L)
    Na 0.2
    Na2•EDTA
    3.73
    Fe 0.1
    FeSO4•7H2O
    2.78
    c.  Micronutrients (100x)
    μM (1x MS)
    Salt
    100x stock (g/L)
    100.0
    H3BO3
    0.62
    100.0
    MnSO4•H2O
    1.69
    30.0
    ZnSO4•7H2O
    0.86
    5.0
    KI
    0.083
    1.0
    Na2MoO4•2H2O
    0.025
    0.1
    Na2MoO4•2H2O
    0.0025
    0.1
    CoCl2•6H2O
    0.0025
    d.  0.5 M CaCl2
    Make the media
    a.  For 1/2 MS 1 L, add:
    50 ml macronutrient 10x stock
    5 ml Fe-EDTA 100x stock
    5 ml micronutrient 100x stock
    Proper CaCl2 0.5 M (add KCl to Ca medium as control for Cl-)
    0.5 g MES (always use 0.5 g for 1 L medium regardless of MS strength)
    Adjust pH to 5.7, add agar to 1% or 0.8%
    Autoclave for 20-30 min
    b.  Composition of plant media (mg/L, 1x)
    Component
    Gamborg’s B5
    Murashige and Skoog
    Total weight
    3,300
    4,620
    Inorganic salts


    CaCl2 anhydrous
    113.23
    332.16
    CoCl2•6H2O
    0.025
    0.025
    CuSO4•5H2O
    0.025
    0.025
    FeSO4•7H2O
    27.8
    27.8
    H3BO3
    3
    6.2
    KH2PO4

    170
    KI
    0.75
    0.83
    KNO3
    2,500
    1,900
    MgSO4•7H2O
    246
    370
    MnSO4•H2O
    10
    16.9
    NaH2PO4•H2O
    150

    Na2-EDTA

    37.3
    Na2MoO4•2H2O
    0.25
    0.25
    NH4NO3

    1,650
    (NH4)2NO3
    134

    ZnSO4•7H2O
    2
    8.6
    Vitamins


    i-Inositol
    10
    100
    Nicotinic acid
    1

    Pantothenic acid.Ca-salt
    0.874

    Pyridoxine•HCl
    1

    Riboflavine
    0.015

    Thiamine•HCl
    10
    0.4*
    * The original formulation contains 0.1 mg/L thiamine.HCl

References

  1. Hoagland, D. R. and Arnon, D. I. (1950). The water culture method for growing plants without soil. California Agric Exp Stn Circ 347: 1-32.

简介

拟南芥作为高等植物的模式生物在世界各地的植物生物实验室中得到广泛研究。 然而,照顾这个小植物可能不是微不足道。 这里是马里兰大学公园的Heven Sze实验室的一般性指南。 Sze实验室和实验室成员已经做了很多努力,为实验室拟南芥植物生长制定了一般指南。

材料和试剂

  1. 拟南芥种子
  2. Miracle-Gro 与肥料的盆栽混合物(2立方英尺袋)(Scotts Miracle-Gro公司,型号:74278300,来自Lowe's,产品编号:156581)
  3. Miracle-Gro ® 8 Qt。 Miracle Gro Perlite(The Scotts Miracle-Gro Company,型号:70752300,Lowe's,产品编号:68468)
  4. 0.1%Triton X-100
  5. 去离子水(去离子水)
  6. 无菌D. 水
  7. 70%乙醇
  8. Drierite
  9. KNO <3>
  10. FeNa-EDTA
  11. KH sub 2 PO 4
  12. MgSO 4
  13. Ca(NO 3)2 CH 4 CH 2 O
  14. 灭菌溶液(参见配方)
  15. 1/4力量Hoagland的解决方案(见配方)
  16. 拟南芥植物在不同Ca 2+/sup>/Mn 2+ 2/条件下的自制MS(Murashige和Skoog,1962) (参见食谱)

设备

  1. 苏格兰奇迹公司
  2. 高压灭菌箱
  3. Scooper
  4. 费舍尔菜
  5. 盆(14 x 8.5 x 6厘米)
  6. 铝箔
  7. 潮湿
  8. 密封容器
  9. 尼龙网
  10. 高压灭菌胶带
  11. 生长室
  12. 微量离心机
  13. 小Fisher皿(100 x 15 mm大小)
  14. Whatman过滤纸

程序

  1. 准备种植土壤的土壤
    我们使用Miracle-Gro ®盆栽与肥料和Miracle-Gro ® 8 Qt混合。 Miracle Gro ®珍珠岩。 为了制作拟南芥土壤,在用于存储土壤的大容器中混合1袋封装混合物(2 cb ft)和半袋珍珠岩(4 qt)。
    注意:我们总是在把土放入种植盆之前对其进行消毒(见下文)。
    1. 将干燥的土壤舀入高压灭菌箱中。填充,直到土壤离开顶部大约一英寸包装下来与打勺。
    2. 用铝箔盖住箱子,并在每个箱子上放一片高压釜胶带。在放入高压灭菌器之前,确保料箱的外部没有污垢。
    3. 在正常/闪烁/快速模式下高压灭菌30-50分钟,具体取决于正在高压灭菌的箱数。
    4. 一旦冷却,土壤可以放入锅中,轻轻地包装。记住要再次覆盖高压灭菌的土壤,以防止任何种子/真菌污染。一旦打开,高压灭菌土壤可以很好地使用长达2周。请不要高压灭菌。
    5. 用去离子水填充平底锅(不在土壤顶部),盖上湿球,并浸泡3-6小时或过夜。浸泡后应有积水(约1英寸),如果没有则加入更多的水。
    6. 盆中的土壤现在准备种植植物。

  2. 种子灭菌
    1. 方法A
      1. 将一定量的种子(野生型,突变体或补充系)放入eppendorf管中。 加入1ml灭菌溶液。
      2. 以最大速度涡旋20-30秒。
      3. 站立7-10分钟,偶尔有涡流。
      4. 在微量离心机中以8,000rpm旋转5秒。
      5. 小心地倒掉水。
      6. 加入1ml无菌水并涡旋以悬浮蓬松的种子。
      7. 在微量离心机中以8,000rpm旋转5秒并倒出上清液。
      8. 重复步骤6-7另外4次。
      9. 将无菌种子移至小的Fisher皿,在其底部用湿的Whatman滤纸(无菌或高压灭菌)。
      10. 用镊子将种子转移到琼脂培养基平板或土壤中,镊子已浸泡在70%乙醇中> 30分钟。
    2. 方法B
      1. 将一定量的种子(不超过0)放入1.5ml Eppendorf管中。 加入1ml 70%乙醇。
      2. 倒出水,加入5 ml 70%乙醇,孵育5分钟。
      3. 用无菌D.I.冲洗。 水4次。
      4. 加入0.1%Triton X-100(或30%Bleach + 0.1%Triton X-100)10分钟。
      5. 用无菌D.I.彻底冲洗。 水(至少5次)。
      6. 将无菌种子移到小Fisher菜,底部有湿的Whatman滤纸。

  3. 种植
    1. 一般关注
      请记住,当它们在亲本植物上生产时,您的种子可能被真菌和细菌污染,对所有植物推荐种子灭菌(直到螺栓时间),并且对于所有产花/种子收集植物和来自其他的种子是必需的 实验室。 室污染通常来自实验室中的脏种子(因为我们总是使用高压灭菌的土壤)。 花部分对昆虫和害虫非常有吸引力,请注意花
    2. 种植健康的拟南芥植物
      在开始之前,托盘必须清洁/擦去没有旧种子或真菌
      1. 用高压灭菌的土壤填充罐(14×8.5×6cm)到顶部。
      2. 添加D.I. 水到包含锅的托盘,约1/3-1/2的锅高。 通常需要至少3小时让土壤彻底浸泡。 然后用喷雾瓶(D.I.水)喷洒土壤表面,以确保种子落在潮湿的表面上。
      3. 将种子转移到土壤(8-10个种子/盆,分开空间种子)。
      4. 种植后,用湿润器覆盖托盘,将其贴在托盘上,以保持必要的湿度。
      5. 将托盘放在4℃的冷室3-5天(5天更好。)在黑暗中。
      6. 移动到生长室与保湿罩仍然覆盖。
      7. 植物应在3-4天后在具有长日光循环的室中发芽。 大约5-7天,当2片子叶已经完全发育时,将潮湿气球半开放为花药2-3天。
      8. 用去离子水除去湿润剂和水。 每周两次(例如星期二和星期五)。

笔记

  1. 拟南芥对所有胁迫条件都很敏感,因此,只有在托盘中没有积水时,才能加水。
  2. 如果植物需要进行开花,每隔一周一次,用1/4强度的Hoagland溶液的水,直到它们开始抽苔(总共两次是足够的)。 Hoagland是促进向生殖生长过渡的富氮培养基。
  3. Miracle-Gro盆栽土壤具有Micracle-Gro植物食物(缓慢释放的肥料),其通常支持植物生长长达3个月。所以抑制了过度施肥的冲动。
  4. 当植物开始开花时注意。根据需要使用任何可能的害虫控制。
  5. 蚜虫是一个常见的昆虫问题。在感染的植物上在罩下喷洒1:100"Orthene",重复喷雾3天。小心不要过度喷洒它们,否则会杀死植物。
  6. 当植物停止开花时,将它们移出到通风的区域以使种子干燥。一旦长角果看起来黄色或棕色,立即收集种子,用很少或没有施加压力打破。
  7. 首先,你应该清理一些区域免费种子的其他物种,以确保所有的种子在这里没有污染与你不想要的其他种子。 用手指在长方形周围擦拭,让种子落在下面的一张大纸上。 将种子通过尼龙网过滤到另一张纸上。 将种子储存在Eppendorf管中,在管的帽上戳一个孔。 收集的种子应在室温下再放置一周以上干燥。 然后将它们放在带有Drierite的密封容器中

食谱

  1. 灭菌溶液
    20%Chlorox漂白剂
    0.05%吐温20 在无菌水中,必须新鲜制备
  2. 1/4强度Hoagland溶液(用于水拟南芥植物)

    FW
    库存
    g/250ml原料
    最后
    ml原料/4L final
    KNO 3
    101.11
    2 M
    50.6
    1.25 mM
    2.5
    FeNa-EDTA
    367.1 20 mM
    1.8355
    12.5μM
    KH 2 PO 4
    136.1
    1 M
    34.0
    0.5 mM
    2.0
    MgSO 4 4 / 120.4
    2 M
    60.2
    0.5 mM
    1.0
    Ca(NO 3)2 2·4H 2 O *
    236.2
    2 M
    118.1
    0.5 mM
    1.0
    未成年人




    1.0
    注意:*当音量已满时,添加最后一个。
    化学品
    FW
    库存(mM)
    最终(μM)
    g/100 ml股票
    H 3 BO 3
    61.83
    70
    17.5


    注意: 从> 3.9升水开始,
    Ca 2 + 可能与SO 4 2-沉淀。
  3. 在不同Ca <2> /Mn 2 + 条件下用于拟南芥植物的自制MS培养基
    库存:
    a。  大量营养素(10x)
    mM(1x MS)
    食盐
    10x原料(g/L)
    41.2
    NH 4 3
    16.5
    18.8
    KNO 3
    19
    3.0
    CaCl 2·无水*
    3.3
    1.5
    MgSO 4·7H 2 O·h/v 3.7
    1.25
    KH 2 PO 4
    1.7
    *对于Ca相关介质,CaCl 2 被排出 b。  Fe-EDTA(100x)
    mM(1x MS)
    食盐
    100x原料(g/L)
    Na 0.2
    Na •EDTA
    3.73
    Fe 0.1
    FeSO 4·7H 2 O·m/2 2.78
    c。  微量营养素(100x)
    μM(1x MS)
    食盐
    100x原料(g/L)
    100.0
    H 3 BO 3
    0.62
    100.0
    MnSO 4 H·H 2 O·m/2 1.69
    30.0
    ZnSO 4·7H 2 O·n/2 0.86
    5.0
    KI
    0.083
    1.0
    Na 2 MoO 4 2H 2 O 0.025
    0.1
    Na 2 MoO 4 2H 2 O 0.0025
    0.1
    CoCl 2 <6h> 6H 2 O
    0.0025
    d。 0.5M CaCl 2/
    制作媒体
    a。  对于1/2 MS 1 L,请添加:
    50 ml宏量营养素10x股票
    5ml Fe-EDTA 100×储液
    5毫升微量营养素100x储液罐
    适当的CaCl 2 0.5M(加入KCl至Ca介质作为Cl - 的对照)
    0.5g MES(对于1L培养基,总是使用0.5g,不考虑MS强度)
    将pH调节至5.7,将琼脂加至1%或0.8%
    高压灭菌20-30分钟
    b。  植物培养基的组成(mg/L,1x)
    组件
    Gamborg's B5
    Murashige和Skoog
    总重量
    3,300
    4,620
    无机盐


    CaCl 2 2水溶液
    113.23
    332.16
    CoCl 2 <6h> 6H 2 O
    0.025
    0.025
    5H2O
    0.025
    0.025
    FeSO 4·7H 2 O·m/2 27.8
    27.8
    > > 4 •7H 2 O
    246
    370
    MnSO 4 H·H 2 O·m/2 10
    16.9
    NaH 2 2 PO 4 4·H 2 O 2···· 150

    > style ="font-size:10.0pt; font-family:""> Na 2 MoO 4 •2H < O
    0.25
    0.25
    NH 4 3

    1,650
    (NH4) 2 NO 3
    134

    ZnSO 4·7H 2 O·n/2 2
    8.6
    维生素


    i-Inositol
    10
    100
    烟酸
    1

    泛酸 0.874

    吡哆醇·HCl
    1

    核黄素
    0.015

    硫胺素·HCl
    10
    0.4 *
    *原始制剂含有0.1mg/L硫胺素

参考文献

  1. Hoagland,D.R。和Arnon,D.I。(1950)。 用于生长没有土壤的植物的水培养方法。 California Agric Exp Stn Circ 347:1-32。
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引用:Li, X. (2011). Arabidopsis Growing Protocol – A General Guide. Bio-protocol Bio101: e126. DOI: 10.21769/BioProtoc.126;
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Maureen McColgin
Wabash College
Can Arabidopsis bolt more than once? I have a lab where I need them newly bolted and they are bolting too early. Any suggestions as to what I can do?
3/25/2012 1:24:33 AM Reply