Something to keep me busy in the winter. I just typed up my notes and I’m just going to paste them in here to share with you.
Chromosome Doubling Experiment, part 3, December 2011
A brief review of my previous attempts:
My theory is that oryzalin could be absorbed through the cut surface of a stem and into a nearby emerging bud. (based on something I read in one of the tissue culture chromosome doubling studies.) A growing shoot is cut off just behind a leaf leaving a section of stem, the plant is allowed to grow until the buds in the apex of the leaves start to swell, and then the stem is trimmed again just above the bud, as close as possible without damaging the bud. The cut surface is quickly moistened (ideally, perhaps, the cut would be made underwater) and the oryzalin solution is applied.
My first attempt was done on roses growing in pots in the greenhouse, approximately in September. I used either a 5 or 10 micromolar solution of oryzalin, and a very small percentage of DMSO (I had intended to use 1.5% DMSO, but mixed up my decimals and ended up with approximately 1/10th of that concentration. Also in the solution was a commercial spreader/sticker wetting agent mixed according to directions; it seemed to be very dilute but apparently the stuff was quite strong. I mixed, about a tablespoon plus two teaspoons of cornstarch into 500 mL of water and microwaved it to create a thicker liquid base. The above ingredients were added to that syrupy cornstarch and water solution. I then wet cotton balls and applied them to the cut surface of the rose stems, holding them in place and wrapping them with tinfoil. (This was a very gooey, nasty, and toxic procedure. I wore a raincoat and three pairs of disposable gloves and changed them often. Make sure you read about the dangers of DMSO if you ever try this.) I left them on for 24 or 48 hours.
That experiment did not result in any apparent tetraploid shoots. If I remember correctly not much happened and I figured the concentration should be stronger or else there should be more DMSO. Unfortunately I didn’t write down the procedure and the details are fuzzy, both for the first and second attempts.
The second attempt was using the same procedure and cornstarch solution. I used about 10 microM oryzalin with 1% DMSO , I think. Left the tinfoil on for 48 hours, including during some fairly warm temps in the greenhouse. In this case I noticed some toxicity, which I was hoping for. Many of the trimmed ends died back. I remember that all of the trimmed ends on Carefree Celebration died. Many of the shoots that survived were killed off when the plants were allowed to dry out at a later date, so this experiment also provided no potentially doubled shoots.
The varieties used in these attempts were: Knock Out, Carefree Celebration, Red Drift, Grouse, Oso Easy Candy Pink, Candy Oh! Vivid Red.
Third try:
On Dec. 16th, I trimmed the shoots on about 5 roses in 4†pots that were cuttings that Betsy Van der Hoek gave me. The variety is likely a thornless Buck understock that had rooted very readily. It sends out long, vigorous horizontal shoots, making it easy to use for the treatment procedure. I treated a total of twelve shoots from those plants, as well as five shoots on Grouse and three shoots on Oso Easy Candy Pink. I waited about four days before treating the plants, growing them at about 72 degrees. I observed that many of the buds had started forming leaves, thus going beyond the bud swell stage that I had desired. Grouse was still in the swelling bud stage, while Candy Pink had shoots about 1/2†long forming leaves. The Buck understock roses varied in how far the buds had emerged, but some of them were up to 1/2†as well.
This time I skipped the cornstarch and used a liquid solution. I purchased water tubes from a florist, the same tubes that are used for a single stem rose. I filled a rectangular container with peat and used the peat to hold the tubes in place. The rose pots were either buried in the peat or lined up along the edge of the container so that the shoots could be bent down into the tubes. I did not attempt to use the covers on the tubes.
I attempted a 5 micromolar solution of oryzalin ( a 1:280,000 dilution according to something I read on the internet). I used a spreader sticker and about 1% DMSO. To make the oryzalin dilution, I used a 1:280 stock solution that I had prepared previously and kept in the fridge. I used a veterinary syringe purchased at a farm supply store to measure .5 mm of this solution into 500 mL water, plus 5 mL DMSO and a surfactant. I used a larger syringe to measure the DMSO and another identical syringe to put the final solution into the water tubes. Most shoots were soaked for 24 hours, and a few for about 38 hours. Nearly all of the shoots ended up with one or two additional nodes (I removed the leaves) immersed in the solution besides the node on the very end. On a few shoots I sliced into the stem near these lower buds in hopes that the oryzalin would soak in through the cut.
David Zlesak had mentioned lowering the temperature before an attempted conversion (he mentioned about 60 degrees) so that the growth tips would accumulate more cells at the proper stage. Therefore I took the plants out of their growing chamber for the duration of the treatment. The temperature ended up between 52 and 57 degrees, probably on the lower end. Not sure if that was too low of a temperature.
It is now about a week after the treatment. Many of the terminal buds, which looked fine right after the treatment, have dried up and died. I don’t know if the drying out and death of those buds is from the oryzalin itself, from the DMSO (which a couple of people have mentioned being phytotoxic,) or simply from the cut being too close to the bud and drying out after it was exposed to air. Enrique, I believe, mentioned on the forum using petroleum jelly to cover the buds after treatment to prevent drying out. It would also be fun to do a comparison trial of clear water (plus surfactant), oryzalin only, DMSO only, and oryzalin + DMSO.
Some of the buds look half dead, and it is those that I will continue to watch in hopes of a conversion. Some stems that were trimmed but not treated show greater bud expansion than the treated stems, which gives me hope that the chemicals did something.