I finished my experiment RWC with 12-day-old transgenic soybean in normal treatment (watering). There was nothing wrong with sample collection, all plant was not dirty and I cleaned out the water left with tissue paper before the FRESH WEIGHT. After that, I sink deeply all plants in distilled water for 24 hours. And then, the TURGID WEIGHT was larger than FRESH WEIGHT, resulted that the RWC was larger than 100% (110-230%). I used the same electrical scale for this experiment.
I'm wondering why this can happen and I don't know why?
I think Turgid weight is supposed to be higher than fresh weight. At least it is according to this protocol document I found with a quick google search.
RWC = ((TFW-BW)-DW)/(TW-DW)
This is not my field but it seems reasonable to me that when you soak plants in distilled water for 24 hours that they will absorb more water.
Your values of RWC seem incorrect. You may want to check your calculations and verify the formula used is correct.
The formula I have used is:
RWC (%) = [(W-DW) / (TW-DW)] x 100
Where,
W – Sample fresh weight
TW – Sample turgid weight
DW – Sample dry weight.
http://www.plantstress.com/methods/RWC.htm
You seem to have used whole plants. Typically, RWC is measured using whole leaves or leaf discs.
Hydration method affects the accuracy of the measurement. Sometimes, it is recommended to do a preliminary study to determine optimal hydration time. To reduce sources of error, hydration may be done in the dark under constant temperature conditions.
I suggest to review the books on Plant Water Relations by Kramer, Weatherley, or Kozlowsky. Those books have sections on RWC measurement.
Good luck!
Agree with Juan. Probably, if you are using the correct formula, you can double check the plant material for the measurement. It is common to have problems with DW. Try increasing the mass of the plant material.
Yes, I used the formula: RWC = (FW-DW)/(TW-DW) * 100
My sample size was n = 80 and all of them have RWC larger than 100 due to Fresh Weight was larger than Turgid Weight. My soybean 12-day-old only has 2 leafs. I divided the shoot and root separately before sank them into distilled water. Hmm, I will think about the hydration as ur suggestion.
Actually, my companions also do this experiment (with plants under drought stress) and their RWC resulted well (70-80%). I followed them but this result makes me really confusing.
Thank you for your guidances. I will do this experiment again. Hope that it will work well ~
Hoa Thi Khanh Tran:
If your FW is higher than your TW it probably means that you are not reaching full hydration of the vegetative material. Try re-cutting the stems (remove a few millimeters of old stem tissue) before you do the hydration.
Good luck!
I think it might be that you soaked it in distilled water there will be isotonic that water (salt) content in the environment and inside the cell of the plant then turgidity will follow. Plant cell will not be easily burst for it contains cell wall which is made of cellulose, strong material in plants...
If you soaked the entire plant then you needed to dry them before you made your TW measurement. Maybe the drying process removed more water than you wanted it to.
@ Gebru
Hmm I will think about it, thank for your suggestion.
@ Kurt
Your opinion was the first reason I thought about. I placed the plant on a plate and used a tissue paper to wash a little water left on the plant which can drop on the scale. My problem happened with the first plant when I compare the data of Fresh Weight and Turgid Weight, so I tried to weight the next plant without clean out the water and the result was still smaller than Fresh Weight. It is really weird and I'm stuck now. So many thanks for your help!
I really want to know the reason to solve it, I will collect samples again in the next week and I would like to do it right. It is really waste time to re-do the experiment without knowing how to solve the problem.
Hoa, I would suggest not submerging your sample. Place the end of the stems into water (a vial) and keep in the dark for 12h. This will rehydrate without the uncertainties of totally submerging the leaves. This is the way I have done it and it has always worked. It is also the common methodology from the literature. I was working with spruce branches. What species are you using?
Also, recut the end of the stem under water first as the water column has snapped up when cut dry.
@ Kurt
I'm working with soybean (actually, transgenic soybean). When I deep the plant into the water, I placed it in the lab room, at room temperature, dark room (because there is no class in the summer semester, so after I left, I turn off all the lights and close windows).
In my opinion, there is no problem in your lab. the value depends on your research subject.
There might be humidity difference/variation in you environment other wise there is no problem with you experiment...
@ Yuanqi
Well, I prepared for my "harvesting day" really careful, I thought that everything will be perfect until I dealt with the data. Thank for your supportive comment!
@ Gebru
... Yes !!! I control everything in safe but my data ran out of the line. I will concern all of your guys' ideas for my next "harvesting day". Thank you!
if as you say all the turgid weights were more than the fresh weight then the rwc should be below 100%
fw-dw/tw-dw
fw-fresh weight
dw-dry weight
tw-turgid wt
It is ideal that your turgid weight is higher than your fresh weight, as RWC is calculated by:
[(FW-DW)/(TW-DW)]*100 and expressed in %
where FW is the fresh weight of the sample, DW is the dry weight and TW is the turgid weight. Probably you missed the DW in your calculation.
The answer to your question lies in the integrity of the cell wall. I just finished an experiment with one treatment having RWC > 100% and also high electrolyte leakage. In this case, FW > Sat weight and therefore, an RWC > 100%.
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