Yes, we have had success with a similar technique, but we were using food coloring in water. In some cases, the dye made it through the branches in minutes. We did not measure the pressure gradient, just elevated the upstream end of the dye tank a meter or so above the branch. The rate of flow could be controlled by changing the elevation. To avoid xylem plugging, we dissolved KCl in the water (40mM solution).

We have also had good success in getting detached tree branches to take up water or dye (food color) solutions simply by putting the cut end into the solution, exposing the branches to light, and allowing the leaves to do the work. It did not take long for the dye to move into the leaves. We also infused solutions directly into the xylem of tall trees in the field and detected the solution in leaves within a couple of days. These techniques have been published; I could send you the references.

Feel free to contact me if I can help further.

Hello, I tried using Safranin (cannot remember the solution concentration) for a similar objective on whole young pine trees (P. patula, 10 year old, about 7m height, about 10 cm DBH) for a cut-tree procedure when calibrating sap flow sensors. Safranin didnt work at all, so I tried vegetable dye instead (the one sold for decorating cakes in the supermarket) and worked perfectly. I thought it may be the alcohol; the vegetable dye is soluble in water.

Yes, we have had success with a similar technique, but we were using food coloring in water. In some cases, the dye made it through the branches in minutes. We did not measure the pressure gradient, just elevated the upstream end of the dye tank a meter or so above the branch. The rate of flow could be controlled by changing the elevation. To avoid xylem plugging, we dissolved KCl in the water (40mM solution).

We have also had good success in getting detached tree branches to take up water or dye (food color) solutions simply by putting the cut end into the solution, exposing the branches to light, and allowing the leaves to do the work. It did not take long for the dye to move into the leaves. We also infused solutions directly into the xylem of tall trees in the field and detected the solution in leaves within a couple of days. These techniques have been published; I could send you the references.

Feel free to contact me if I can help further.

We use acid fuchsin, which is soluble in water. The speed of 40 cm/hour is roughly the speed of the xylem sap in the living diffuse porous tree in daytime - it's nothing wrong with it.

I used 10% fuchsine in distilled water by shallow trunk injection method and other injection and infusion methods with no pressure. Due to variable methods dye moves and accumulates variably - but being that the dye is water soluble it is a very good indicator where it accumulates. Usually 10-15 days after introduction of dye we see this variable accumulation - so the speed of uptake you mention is OK. But, to answer your question - use dye that is well soluble in water, and maybe you'll get more uptake and more dye movement by the method you use.

Hi Peter, the slow rate of flow may be because the safranin is a positively charged dye and is adhering to the negatively charged xylem walls. Perhaps try a dye with a negative charge, e.g. the acid fuchsin that Josef suggested. Good luck - it's frustrating because these experiments are time-consuming.

Hi Mary Anne Mcguire, can you attach the references your mentioned here?

Hi Peter

we performed several trials about dye transport along trunk tissues of Palm trees, in particular Phoenix sspp. We used both safranin and methylene blue aqueous solutions, injected into the stem basal part with a needle about 25 cm long. Palms have scattered vascular bundles, and it's therefore not easy to follow the vascular stream. We cut the treated trunks in pieces, to carry out the visual observations. In general, the injection of dye allowed good results, as you can see from the alleged pictures. Herein you can see the hole of needle, the tissue dye within vascular elements (red and blue, of course). Best wishes for you work!!!

We determine flow rate and sapwood area on small diameter branches between 1 and 2 cm in diameter. Initial water flow rate for each segment is measured at 7 .5 kPa from a low-pressure reservoir by reading off the mass (in grams) of the collecting beaker on a four point balance every 30 seconds for 2.5 minutes. The segment is then flushed for 20 minutes at 150 kPa, to remove emboli, and the flow rate re-measured This procedure is repeated until the flow rate stabilizes presumably because all emboli have been removed, giving the maximum flow rate for each segment.

We then fit a 2 m length of clear PVC pipe tightly around one end of the branch. The sapwood in each stem segment is then stained by filling the pipe with a 0.5% safrinin dye, 1OmM KCI solution with degassed de-ionized filtered (0.45 pm membrane filter) water, allowing for gravity flow of the dye through the segment. Cross sections of the stained segments are then photographed and analysed using the Image J analyzing software package to determine sapwood areas.

We determine flow rate and sapwood area on small diameter branches between 1 and 2 cm in diameter. Initial water flow rate for each segment is measured at 7 .5 kPa from a low-pressure reservoir by reading off the mass (in grams) of the collecting beaker on a four point balance every 30 seconds for 2.5 minutes. The segment is then flushed for 20 minutes at 150 kPa, to remove emboli, and the flow rate re-measured This procedure is repeated until the flow rate stabilizes presumably because all emboli have been removed, giving the maximum flow rate for each segment.

We then fit a 2 m length of clear PVC pipe tightly around one end of the branch. The sapwood in each stem segment is then stained by filling the pipe with a 0.5% safrinin dye, 1OmM KCI solution with degassed de-ionized filtered (0.45 pm membrane filter) water, allowing for gravity flow of the dye through the segment. Cross sections of the stained segments are then photographed and analysed using the Image J analyzing software package to determine sapwood areas.

Thank you for all of your responses to my question, these have been most helpful. Edmund, we are doing exactly the same thing, I also use imageJ to make the calculations. I will work on the dye mixture and add KCl. I will also try some of the other dyes recommended. Also try positively charged dyes as suggested by Virginia. Paolo, I am interested to see some of the results with the palm studies. Martin Zimmermann made some movies that traced the monocot vasculature, I am not sure if there is a digital copy available, but if you can find these videos, you must have a look.

References attached as requested. (In these studies we used dye in preliminary tests only to determine the amount of time needed for our main treatments to reach their destination.)

Thanks Mary Anne!

Have you thought to use a vacuum pump to increase the gradient for dye movement through branch? You can attach a vacuum to one extreme of stem and perfusing dye in another.

Good luck

Alberto

Yes Alberto, good idea with the vacuum pump, but I have large branches with all the distal ends excised, making it difficult to use a vacuum pump method, I seem to make things difficult...

We use a 0.1% safranin solution (1g / 1L) disolved in destilled degased water (1mM CaCO3 and 10mM KCl) to color the sapwood area of large stems attached to a vacuum pump (c. 500mbar) used for sapflow sensor calibrations (diameter c. 10cm, length 1m), the method has been described by Bush et al. 2010: Calibration of thermal dissipation sap flow probes for ring- and diffuse-porous trees.

I haven't used this set-up for small-diameter branches used for hydraulic conductivity measurements but assume it should work as fine. Since we flush samples at 1.2 bar (standard protocol I assume) I would have pushed the dye solution at that pressure through the segment if no vacuum pump is available.

Cheers, Bernhard

Hi Peter!

We have long noticed that Safranine is flowing very slowly in the xylem. My experience is that this molecule needs to saturate all the site of fixation on the walls before moving further away. Phloxine moves as fast as the sap flows but also diffuses laterally quite easily. You should also use aquaous solutions.

Hervé