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This protocol establishes growth of Arabidopsis seedlings in a hydroponic medium that is suitable for the controlled addition or withdrawal of any number of nutrients, hormones or metabolites to the plants via the roots. In this case, the described protocol is used to assay sensitivity of shoot branching to exogenously supplied strigolactone, typically the artificial strigolactone GR24. Plants deficient in strigolactone synthesis or response will typically exhibit an increased number of axillary branches compared to wild type. Strigolactone synthesis mutants should respond to exogenous GR24 with reduced numbers and length of axillary shoots, while the phenotype of response mutants will not be rescued.
We present here a more detailed protocol extended from that described in Waters et al. (2012).

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Arabidopsis Hydroponics and Shoot Branching Assay
水培条件下研究拟南芥地上部分枝发育的试验方法

植物科学 > 植物生理学 > 植物生长
作者: Mark T Waters
Mark T WatersAffiliation: ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Perth, Australia
For correspondence: mark.waters@uwa.edu.au
Bio-protocol author page: a109
John D Bussell
John D Bussell Affiliation: ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Perth, Australia
Bio-protocol author page: a110
 and Ricarda Jost
Ricarda JostAffiliation: School of Plant Biology, The University of Western Australia, Perth, Australia
Bio-protocol author page: a111
Vol 2, Iss 19, 10/5/2012, 11890 views, 5 Q&A
DOI: https://doi.org/10.21769/BioProtoc.264

[Abstract] This protocol establishes growth of Arabidopsis seedlings in a hydroponic medium that is suitable for the controlled addition or withdrawal of any number of nutrients, hormones or metabolites to the plants via the roots. In this case, the described protocol is used to assay sensitivity of shoot branching to exogenously supplied strigolactone, typically the artificial strigolactone GR24. Plants deficient in strigolactone synthesis or response will typically exhibit an increased number of axillary branches compared to wild type. Strigolactone synthesis mutants should respond to exogenous GR24 with reduced numbers and length of axillary shoots, while the phenotype of response mutants will not be rescued.
We present here a more detailed protocol extended from that described in Waters et al. (2012).

[Abstract] 该实验方案的中文版正在准备中...

Materials and Reagents

  1. Rock wool/mineral wool (see http://en.wikipedia.org/wiki/Rock_wool; available from hydroponics suppliers)
  2. GR24, 10 mM in 100% acetone, stored in -20 °C freezer (limited availability; can be purchased from Chiralix: http://www.chiralix.com/rightclick.cfm?id=61574)
  3. Acetone
  4. MilliQ water
  5. Ca(NO3)2
  6. KNO3
  7. NH4NO3
  8. MgSO4.7H2O
  9. KH2PO4
  10. KCl
  11. H3BO3
  12. MnCl2.4H2O
  13. ZnSO4.7 H2O
  14. CuSO4.5 H2O
  15. CoCl2.6 H2O
  16. (NH4)6Mo7O24.4 H2O
  17. Fe-EDTA
  18. 2-(N-morpholino) ethanesulfonic acid, a buffering agent (MES)
  19. 0.5x Hoagland’s nutrient solution (pH 5.9) (see Recipes)
  20. Hoagland’s solutions (see Recipes)

Equipment

  1. Controlled environment growth chamber or room
  2. Sealed plastic box for growing plants, with transparent lid and with rack to hold 1.5 ml microcentrifuge tubes , e.g. I5100-43, Astral Scientific, NSW, Australia. Also available from:
    http://www.bioexpress.com/divinity-cart/item/502100/GeneMate-Floater-Microtube-Rack/1.html
    http://www.labsource.com/Catalog/Group.aspx?GroupID=1455
  3. Black 1.5 ml microcentrifuge tubes (opaque tubes minimizes light transmission and hence algal growth)
  4. Fine forceps
  5. Cork borer, 8 mm diameter, to produce cylinders of rock wool (see http://en.wikipedia.org/wiki/Cork_borer)


    Figure 1. Three-week-old Arabidopsis Ler plants grown in hydroponics according to this protocol. The lid of the box has been removed in this photograph. Grid squares are 1 cm.

Procedure

  1. Plan how many plants you will need for the experiment. If you use the plastic box suggested above, you can comfortably fit 4 adult plants in a single pot (see Figure 1). If each pot counts as a replicate, and you want four biological replicates, then you will need 4 pots (16 plants) per genotype per treatment. You need at least as many microcentrifuge tubes as plants, plus some spares (10-20%) to allow for failures of seedlings to grow.
  2. Using scissors or a hot scalpel blade, cut off the cap and the bottom 5 mm (or so) from each microcentrifuge tube.
  3. Cut a slice of rock wool about 2 cm in height, saturate with tap water, and place in a suitable plastic dish.
  4. Using the cork borer, very gently punch out a cylinder of wet rock wool. This takes some practice. Push down with very little force, while rotating the puncher left and right to help it work its way down. Often the cylinder will be trapped within the borer; use a suitable implement to gently push it out from the top of the borer. It is important that the cylinders are not crushed, so that the seedling’s root can penetrate the matrix easily (see Figure 2A, B).


    Figure 2. Setting up the rock wool plugs. A. Pushing gently and with a rotating motion, cut a series of cylinders from saturated rock wool using an 8 mm cork borer. When you reach the bottom, push firmly to cut the cylinder free. B. Use a plastic tool to gently push out the cylinder from the borer. C. Place each cylinder in a black microcentrifuge tube, such that the rock wool is level with the top of the tube. D. Place the tubes in the rack. You can fill every hole at this stage.

  5. Make as many rock wool cylinders as are required for the number of microcentrifuge tubes prepared in step 2.
  6. Using forceps, place the cylinders in each tube, and gently press into place so that the cylinder is flush with the top of the tube. Again, do not compress the rock wool (see Figure 2C. It doesn’t matter if the rock wool sticks above the top edge of the tube by a few millimetres, but the closer to the bottom of the tube it is, the less solution you will need to keep it wet).
  7. Dilute some 0.5x Hoagland’s nutrient solution 1 in 2 (i.e. 0.25x), place sufficient in the base of the growth box to come up to the bottom of the rack, and fill the rack’s holes with the tubes containing rock wool. During early growth establishment, all of the positions in the rack can be filled with tubes. Later on, when the plants mature, the tubes will need to be split between multiple racks (see step 13). At this point the tubes and rockwool may be autoclaved, but this is optional.
  8. Wet the tip of some fine forceps, and then pick up about 5-10 Arabidopsis seed. Deposit the seed on the surface of the rock wool, and repeat for all tubes.
  9. Close the lid on the box and stratify the seed at 4 °C for three days.
  10. Transfer the box to a growth room with approx. 100-200 μmol photons m-2 sec-1 light intensity, 21 °C, long days (16 h light) for fast growth. Keep the lid closed.
  11. After about three days, lift the lid gradually over the next three days or so. We use a 1 ml pipette tip to prop the lid open by about 1 cm on the first day, moving it backwards gradually to open the lid further. This helps to protect the seedlings from shock to the drier atmosphere. Check that the nutrient solution is still in contact with the bottom of the tubes; if they dry out, the seedlings will die very quickly! Top up with 0.25x Hoagland’s solution if necessary.
  12. When the seedlings are seven days old, open the lid fully, and thin out the seedlings to 1 per tube, using fine forceps. At this point, replace the nutrient solution with 0.5x Hoagland’s, and commence the GR24 treatment by supplementing with the required volume of GR24 (5 μM final concentration) or acetone for untreated controls. It is suggested that you add the hormone/acetone to the solution in a bottle, mix well and then dispense the required volume into the boxes.
  13. After about 10-12 days, the roots should be coming out of the bottom of the tubes and floating in the medium. At this point, before the roots get too long, split the seedlings across as many boxes as required for your experiment, bearing in mind how much space each plant needs. Dispose of any excess plants (but keep the tubes for any future experiments).
  14. Allow the plants to grow, keeping an eye on the nutrient level. There is a trade-off between time spent looking after the plants, GR24 hormone consumed, and frequency of nutrient exchange. I suggest in the first two weeks of growth, changing the solution every 5 days or so. After three weeks, the plants consume more nutrients, so the solution will need exchanging every 3 to 4 days, depending on humidity. Stability of GR24 is another issue; it is prone to hydrolysis and a very approximate (and generous) guess would be that it has a half-life of 1 week under these conditions. Do not “top up” the solution; replace it fully with fresh solution plus GR24 as required.
  15. When the plants begin to flower, there is no longer any need to continue the GR24 treatment, as all rosette leaves will have been formed. At this point, count the number of rosette leaves, should you want to express numbers of branches per rosette leaf.
  16. When the primary inflorescence stops producing flowers, it is time to count the number of axillary branches and, if desired, the height of the main inflorescence stem. We normally count an axillary branch if it has grown more than 5 mm.

Expected Outcome

  1. For mutants that are deficient in the synthesis or levels of endogenous strigolactones, the number of axillary branches should be reduced relative to untreated controls. For strigolactone insensitive mutants, the number of branches should not be significantly affected. Wild type plants normally do not show a significant reduction in branch number, as it is normally low anyway (1-2 branches, depending on ecotype and growth conditions). In our hands, this method does not lead to complete rescue of the strigolactone-deficient mutant phenotype to wild type levels, but does noticeably reduce branching.

Recipes

  1. 0.5 x Hoagland’s nutrient solution (pH 5.9)
    Modified from Heeg et al. (2008)



  2. Hoagland’s solutions
    Use MilliQ water to prepare, store Solutions I and II at room temperature and all others at 4 °C.
    Autoclave all solutions (EXCEPT Solution I) using a cycle with 15 min at 121 °C.
    Stock solution I (500x): 1 M Ca(NO3)2.4H2O 236.2 g/L, filter-sterilise
    Stock solution II (500x): 1 M KNO3 101.1 g/L, autoclave
    Stock solution III (1,000x): 0.5 M NH4NO3 40.0 g/L, autoclave
    Stock solution IV (500x): 0.25 M MgSO4.7H2O 61.6 g/L, autoclave
    Stock solution V (1,000x): 0.25 M KH2PO4 34.0 g/L, autoclave
    Stock solution VI (5,000x): 0.25 M KCl 18.6 g/L, autoclave
    Micro element stock solution (2,000x):
    0.05 M H3BO3 1.546 g
    0.004 M MnCl2.4 H2O 0.396 g
    0.004 M ZnSO4.7 H2O 0.575 g
    0.001 M CuSO4.5 H2O 0.125 g
    0.0003 M CoCl2.6 H2O 0.036 g
    0.00015 M (NH4)6Mo7O24.4 H2O 0.093 g
    Add 500 ml ddH2O, autoclave.
    10 mM Fe-EDTA stock soln. (250x): 367.1 mg/100ml, autoclave.
    Check pH if it is not dissolving and adjust pH to 8.0 using 5 N KOH or 1 N NaOH (if you want to get some Na+ ions into your solution).
    For 10 L of Hoagland’s solution:
    Dissolve 5 g MES in 8 L milliQ water and add, while mixing:
    Stock solution I 20 ml
    Stock solution II 20 ml
    Stock solution III 10 ml
    Stock solution IV 20 ml
    Stock solution V 10 ml
    Stock solution VI 2 ml
    Micro elements 5 ml
    Fe-EDTA 40 ml
    Adjust the pH of the solution with 5 N KOH to pH 5.8-6.0. THIS IS CRITICAL: GR24 is increasingly unstable at low or high pH. Make up volume up to 10 L.

Acknowledgements

We are grateful to Dr Brent Kaiser (University of Adelaide, Australia) for providing us with the hydroponics method outlined in this protocol. This protocol was further developed from Hoagland and Arnon (1941). The water culture method for growing plants without soil was adapted from miscellaneous publications including Gibeaut et al. (1997) and Tocquin et al. (2003). The work was funded by grants from the Australian Research Council (LP0776252 (RJ) and DP1096717 (MW)).

References

  1. Gibeaut, D. M., Hulett, J., Cramer, G. R. and Seemann, J. R. (1997). Maximal biomass of Arabidopsis thaliana using a simple, low-maintenance hydroponic method and favorable environmental conditions. Plant Physiol 115(2): 317-319.
  2. Heeg, C., Kruse, C., Jost, R., Gutensohn, M., Ruppert, T., Wirtz, M. and Hell, R. (2008). Analysis of the Arabidopsis O-acetylserine(thiol)lyase gene family demonstrates compartment-specific differences in the regulation of cysteine synthesis. Plant Cell 20(1): 168-185.
  3. Hoagland, D. R. and Arnon, D. I. (1941): The water culture method for growing plants without soil. Miscellaneous Publications 354: 347-461.
  4. Waters, M. T., Nelson, D. C., Scaffidi, A., Flematti, G. R., Sun, Y. K., Dixon, K. W. and Smith, S. M. (2012). Specialisation within the DWARF14 protein family confers distinct responses to karrikins and strigolactones in Arabidopsis. Development 139(7): 1285-1295.
  5. Tocquin, P., Corbesier, L., Havelange, A., Pieltain, A., Kurtem, E., Bernier, G. and Perilleux, C. (2003). A novel high efficiency, low maintenance, hydroponic system for synchronous growth and flowering of Arabidopsis thaliana. BMC Plant Biol 3: 2.

材料和试剂

  1. 岩棉/矿棉(请参阅 http://en.wikipedia.org/wiki/Rock_wool ; 可从水栽法供应商获得)
  2. GR24,10mM在100%丙酮中,储存在-20℃冰箱中(有限的可用性;可购自Chiralix: http://www.chiralix.com/rightclick.cfm?id=61574
  3. 丙酮
  4. MilliQ水
  5. Ca(NO 3)/2。
  6. KNO 3
  7. NH 4 3
  8. MgSO 4。 。 O
  9. KH 2 PO 4
  10. KCl
  11. H 3 BO 3
  12. MnCl 2 4H O
  13. ZnSO 4 7 H O
  14. CuSO 4 5 H O
  15. CoCl <2> 6 H <2> O
  16. (NH 4)6 Mo 7 SubO 24。 2 O
  17. Fe-EDTA
  18. 2-(N-吗啉代)乙磺酸,缓冲剂(MES)
  19. 0.5x Hoagland营养液(pH5.9)(见配方)
  20. Hoagland的解决方案(参见配方)

设备

  1. 受控环境生长室或房间
  2. 用于生长植物的密封塑料盒,具有透明盖并且具有保持1.5ml微量离心管,例如澳大利亚新南威尔士州Astral科学公司的I5100-43的微量离心管。 也可从:
    http://www.bioexpress.com /divinity-cart/item/502100/GeneMate-Floater-Microtube-Rack/1.html
    http://www.labsource.com/Catalog/Group.aspx?GroupID=1455
  3. 黑色1.5ml微量离心管(不透明管使光传播最小化,因此藻类生长)
  4. 细镊子
  5. 软木bore,直径8毫米,用于生产岩棉圆柱(见 http://en.wikipedia.org/wiki/Cork_borer


    图1。 三周龄 拟南芥 使根据本协议在水培法中种植的植物。 盒子的盖子已在此照片中删除。 网格正方形为1厘米。

程序

  1. 计划实验需要多少植物。如果你使用上面建议的塑料盒,你可以舒适地适合4成年植物在一个锅(见图1)。如果每个罐计为一个重复,并且你想要四个生物复制,那么你将需要每个处理4个盆(16植物)每基因型。你至少需要与植物一样多的微量离心管,加上一些备件(10-20%)以允许苗的生长失败。
  2. 使用剪刀或热刀手术刀,切下帽和底部,每个微量离心管5 mm(或以上)。
  3. 切一块高约2厘米的岩棉,用自来水浸透,放入合适的塑料盘。
  4. 使用软木钻孔器,非常轻轻地打出一个湿的岩棉缸。这需要一些实践。用非常小的力向下推,同时向左和向右旋转打孔机以帮助它向下工作。通常气缸将被困在钻孔器中;使用合适的工具轻轻地将其从钻孔器的顶部推出。重要的是,圆筒不被压碎,使得苗的根部可以容易地穿透基体(参见图2A,B)。


    图2。 设置岩棉塞。A.轻轻地旋转,用8毫米软木钻头从饱和岩棉切割一系列圆柱体。当您到达底部时,用力推动气缸自由切割。 B.使用塑料工具轻轻地将圆筒从钻孔器中推出。 C.将每个圆筒放在黑色的微量离心管中,使岩棉与管的顶部齐平。 D.将管放入 架。你可以在这个阶段填充每个洞。

  5. 制作与步骤2中准备的微量离心管数量一样多的岩棉滚筒。
  6. 使用镊子,将气瓶放在每个管中,轻轻压入到位,使气瓶与管的顶部齐平。再次,不要压缩岩棉(见图2C。无论岩棉如何粘在管子顶部边缘上方几毫米,而是越靠近管子底部,越少的解决方案你需要保持湿)。
  7. 稀释0.5x Hoagland的营养液1(2)( 0.25x),放在生长箱底部足够到达架子底部,并用管子填满架子的孔含石岩棉。在早期生长建立期间,机架中的所有位置可以填充有管。稍后,当植物成熟时,管将需要在多个架之间分开(参见步骤13)。在这一点上,管和岩棉可以被高压灭菌,但这是可选的
  8. 润湿一些细镊子的尖端,然后拾取约5-10个拟南芥种子。将种子存放在岩棉表面,并对所有管重复。
  9. 关闭盒子上的盖子,并在4℃下将种子分层三天
  10. 将盒子转移到一个大约的成长室。 100-200μmol光子m-2秒-1光强度,21℃,长天(16小时光),用于快速生长。保持盖子关闭。
  11. 大约三天后,在接下来的三天左右逐渐提起盖子。我们使用1毫升移液器提示在第一天支撑盖子打开约1厘米,逐渐向后移动,以进一步打开盖子。这有助于保护幼苗免受干燥气氛的冲击。检查营养液是否仍然与管的底部接触;如果他们干了,幼苗会死的很快!如有必要,加上0.25x Hoagland的解决方案。
  12. 当幼苗是七天大时,完全打开盖子,使用细镊子将幼苗稀释至每管1个。此时,用0.5x Hoagland's替换营养液,并通过补充所需体积的GR24(5μM终浓度)或丙酮用于未处理的对照开始GR24处理。建议您在瓶中的溶液中加入激素/丙酮,充分混合,然后将所需体积分配到盒子中。
  13. 约10-12天后,根应该从管的底部出来并漂浮在培养基中。在这一点上,在根变得太长之前,将幼苗分成与实验所需的盒子一样多,考虑到每株植物需要多少空间。处理任何多余的植物(但保留试管用于任何未来的实验)
  14. 允许植物生长,保持眼睛的营养水平。在观察植物的时间,消耗的GR24激素和营养物交换频率之间存在权衡。我建议在前两个星期的生长,每5天左右改变解决方案。三个星期后,植物消耗更多的营养,所以解决方案将需要交换每3到4天,这取决于湿度。 GR24的稳定性是另一个问题;它容易水解,并且非常接近(和宽泛)的猜测将是它在这些条件下具有1周的半衰期。不要"加满"解决方案;请根据需要用新鲜溶液和GR24充分更换
  15. 当植物开始花时,不再需要继续GR24处理,因为所有的莲座叶都将形成。在这一点上,计算玫瑰花叶的数量,你应该表示每个玫瑰叶的分支数量。
  16. 当主花序停止产生花时,是时候计数腋枝的数目,如果需要,计数主花序茎的高度。如果生长超过5毫米,我们通常计算腋窝分支

预期结果

  1. 对于内源性独脚金内酯的合成或水平缺乏的突变体,相对于未处理的对照,腋窝分枝的数目应当减少。对于独脚金内酯不敏感突变体,分枝数目不应受到显着影响。野生型植物通常不显示分支数目的显着减少,因为它通常是低的(1-2个分支,取决于生态型和生长条件)。在我们的手中,这种方法不导致完全拯救独脚金内皮缺陷型突变表型到野生型水平,但明显减少分支。

食谱

  1. 0.5×Hoagland营养液(pH5.9)
    从Heeg等人修改。(2008)



  2. Hoagland的解决方案
    使用MilliQ水制备,在室温下储存溶液I和II,在4℃下储存所有其它溶液 使用在121℃下15分钟的循环,高压灭菌所有溶液(EXCEPT Solution I)。
    储备溶液I(500×):1μMCa(NO 3)2+ 236.4g/l 236.2g/L,过滤灭菌
    储备溶液II(500x):1M KNO 3 101.1g/L,高压釜
    储备溶液III(1,000x):0.5M NH 4 NO 3 40.0g/L,高压釜
    储备溶液IV(500×):0.25M MgSO 4。 7H 2 O 61.6g/L,高压釜
    储备溶液V(1,000x):0.25M KH 2 PO 4 4 34.0g/L,高压釜
    储备溶液VI(5,000x):0.25M KCl 18.6g/L,高压釜
    微元素储备溶液(2,000x):
    0.05 M H sub 3 BO 3 1.546 g
    0.004M MnCl 2 4 H 2 O 0.396g
    0.004M ZnSO 4 sub
    H H 2 O 0.575g
    0.001 M CuSO 4 5 H sub 2 O 0.125 g
    0.0003 M CoCl 2 6 H 2 O 0.036 g
    0.00015 M(NH 4)6 Mo 7+ O 24。 H sub> 2 O 0.093 g
    加入500ml ddH 2 O,高压釜 10mM Fe-EDTA储备溶液。 (250×):367.1mg/100ml,高压灭菌 如果不溶解,检查pH值,用5N KOH或1N NaOH调节pH值到8.0(如果你想在溶液中加入一些钠离子)。
    对于10 L Hoagland的解决方案:
    将5g MES溶解在8L milliQ水中,并在混合下加入:
    储备溶液I 20 ml
    储备溶液II 20 ml
    储备溶液III 10 ml
    储备溶液IV 20 ml
    储备溶液V 10 ml
    储备溶液VI 2 ml
    微量元素5 ml
    Fe-EDTA 40ml
    用5N KOH将溶液的pH调节至pH 5.8-6.0。 这是关键:GR24在低或高pH时越来越不稳定。 补偿音量高达10 L.

致谢

我们感谢Brent Kaiser博士(澳大利亚阿德莱德大学)为我们提供了本协议中概述的水培方法。 该方案进一步从Hoagland和Arnon(1941)开发。 用于生长没有土壤的植物的水培养方法来自杂项出版物,包括Gibeaut等人(1997)和Tocquin等人(2003)。 该工作由澳大利亚研究委员会(LP0776252(RJ)和DP1096717(MW))的赠款资助。

参考文献

  1. Gibeaut,D.M.,Hulett,J.,Cramer,G.R.and Seemann,J.R。(1997)。 拟南芥的最大生物量使用简单,低维护的水培法和有利的环境条件。植物生理学 115(2):317-319。
  2. Heeg,C.,Kruse,C.,Jost,R.,Gutensohn,M.,Ruppert,T.,Wirtz,M。和Hell,R。(2008)。 拟南芥O-乙酰丝氨酸(硫醇)裂解酶基因家族的分析证明半胱氨酸合成调节的区室特异性差异。植物细胞 20(1):168-185。
  3. Hoagland,DRand Arnon,DI(1941):用于生长植物的水培养方法 杂项出版物 354:347-461。
  4. Waters,M.T.,Nelson,D.C.,Scaffidi,A.,Flematti,G.R.,Sun,Y.K.,Dixon,K.W.and Smith,S.M。 DWARF14蛋白家族中的专业化对拟南芥中的karrikins和独脚金内酯具有不同的反应。 开发 139(7):1285-1295。
  5. Tocquin,P.,Corbesier,L.,Havelange,A.,Pieltain,A.,Kurtem,E.,Bernier,G。和Perilleux,C。 拟南芥同步生长和开花的新型高效,低维护,水培系统 。 BMC Plant Biol 3:2.
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How to cite this protocol: Waters, M. T., Bussell, J. D. and Jost, R. (2012). Arabidopsis Hydroponics and Shoot Branching Assay. Bio-protocol 2(19): e264. DOI: 10.21769/BioProtoc.264; Full Text



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8/7/2013 2:19:16 PM  

EMRE EMRE AKSOY
Horticultural Sciences Department, Texas A&M University, USA

Mark, did you use any aeration during the growth? How does it affect the growth?

8/7/2013 6:37:09 PM  

Mark Waters (Author)
ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Australia

No: for simplicity and ease of scaling, we did not use or try aeration. The boxes we use measure about 13 cm x 9 cm (LxW) and we add 160 ml of solution to each. That gives a decent surface area:volume ratio, or so it would seem. The solution, when changed every three days or so, either contains sufficient oxygen, or there is enough gas exchange through the other holes in the boxes. I have not formally compared growth rates with those of plants grown on soil or in other hydroponics systems, but they *seem* to grow pretty normally, flower, set seed etc. Probably aeration would help but it does complicate the set up. A maximum of six plants per pot is tolerable, though I prefer to use four to give them a bit more space. Depends in part on how valuable your media/compounds are.

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7/20/2013 3:56:08 AM  

Mayvel Lapuz
University of the Philippines

Sir, I like to know if you monitored the concentration during the duration of the study. If yes, What methods did you use?

7/20/2013 3:56:53 AM  

Mayvel Lapuz
University of the Philippines

I mean the concentration of the substrate.

7/21/2013 9:56:18 PM  

Mark Waters (Author)
ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Australia

The simple answer is no, we did not measure the concentration of GR24 during the study. It is possible to do so by HPLC to monitor degradation. From our tests, the half-life of GR24 at pH5.7 is about 3 days, but we have not explicitly measured its half-life in Hoaglands media.

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12/17/2012 4:24:40 PM  

Mark Waters (Author)
ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Australia

I would make up a 1x Hoagland's solution, by doubling all the volumes of the various component solutions. I would then make up a NaCl solution of twice the required strength, and mix 1:1 with the 1x Hoagland's. So to make various NaCl concentrations, just make several NaCl solutions of 2x the final concentration desired.

e.g. to treat plants with 150 mM NaCl in 0.5x Hoagland's, mix equal volumes of 1x Hoagland's with 300 mM NaCl.

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12/16/2012 6:28:50 PM  

iam aske about how can i mixed Hoagland solution with NaCl at different concetration

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10/15/2012 8:40:09 AM  

I am using your protocol to do phosphate assays, and I am curious: do you make your Hoagland's in lab or do you purchase it? If you purchase it, from where? Thank you.

10/15/2012 10:52:36 PM  

Mark Waters (Author)
ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Australia

We make each of the stock solutions ourselves in the lab. This is potentially expensive if you have to buy all the reagents, especially considering how little you need of some of the micronutrients. Some of the micronutrients can be substituted, e.g. ZnCl2 rather than ZnSO4, as it is the Zn2+ rather than the Cl- or SO4- that matters.

2/5/2013 7:45:09 AM  

Charles Parrish
North Carolina State University

Thank you, Dr. Waters. I would also like to know where you purchased the rockwool used in your experiment, and what type of rockwool it is? Thank you.

2/12/2013 6:27:24 PM  

Mark Waters (Author)
ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Australia

Hi Charles

We get our rock wool from these guys in Australia:

http://www.growroom.com.au/shop/propagation/rockwool-wrapped-slab-75x900x150/

The manufacturer is Grodan, and you may be able to find alocal distributor here:

http://www.grodan.com/about+grodan/find+a+distributor

2/12/2013 6:28:23 PM  

Mark Waters (Author)
ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Australia

And more information on what rock wool actually is:

http://www.grodan.com/about+grodan/stone+wool+substrate

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