I read a PhD dissertation by Ryan Nelson Contreras a while back about inducing polyploidy, among other things, in Japanese cedar (Contreras, 2009). The technique he used was to spray an aqueous solution of oryzalin on Japanese cedar seedlings for 30 days. From the paper: “Seedlings were sprayed to run-off daily for 30 d with an aqueous solution containing 150 μ M oryzalin (supplied as Surflan® AS, United Phosphorus, Trenton, NJ) + 0.1% SilEnergy™ (Brewer International, Vero Beach, Fla.), an organosilicate surfactant, using a standard spray bottle (Model P-32, Sprayco, Farmington Hills, Mich.).” To brush up on your polyploidy knowledge, read my paper “Polyploidy in Plant Breeding.”
The result of this effort was that a high percentage of the seedlings were converted to tetraploids, 83% of the survivors. Around 2/3 of the seedlings didn’t survive (237 survivors out of 600). A side effect of this process are chimaeras, where you have incomplete doubling in the plant tissues, with a mix of diploid and tetraploid cells.
For the plant breeder dealing with large numbers of seedlings, you need to simplify where you can. When making tetraploids, you want your seedlings either dead or converted to make screening easier. After the treatment, it’s another big job to screen your seedlings for polyploids; a visual screen will make the job easier if you can see obvious differences. Dr. Contreras (I assume he’s a Dr. now) found a high correlation between thick, twisted leaves and tetraploidy (92% accuracy).
So after reading this I thought, “Why not try this myself?” I decided to attempt a conversion on various species and hybrids of Passiflora. I chose some P. incarnata seeds that I had collected from the wild, as well as P. edulis, P. tucumanensis, P. umbilicata, P. alata, P. caerulea, P. (arida x sublanceolata), P. sublanceolata, P. ‘Incense’, and P. arida.
I obtained some Surflan, trade name for oryzalin, which is a thick, neon-orange liquid used as a pre-emergent herbicide (diluted, of course). It prevents seeds from germinating by disrupting mitosis. Cells can’t divide and seeds subsequently die. However, in smaller doses it can be used to disrupt mitosis enough to double the chromosomes without a fatality. The trick is finding the right dose and duration of application. The other ingredient, SilEnergy, is a pentrating adjuvant which enhances the effectiveness of herbicides by helping them get into plant tissue. (Adjuvant description) I ordered it from an aquatic herbicide company, from which now I am destined to receive a lifetime supply of catalogues.
These chemicals are safer than some things, but still I would err on the side of caution. If in doubt, you can always look at the MSDS (Material Safety Data Sheet) which is available from the manufacturer. Legally they have to provide it. Here is the MSDS for SilEnergy and here’s the MSDS for Surflan. The basic summation for these two is: don’t eat it, wear gloves. Just be smart; don’t mix it up in the kitchen sink.
The concentration Contreras used was 150µM. I talked to another breeder who used a concentration much lower, which I felt was too low. Again, I want conversion or death. The next step is to determine optimum duration. I decided to start with the same duration: 30 days.
I planted my seeds into four inch pots and waited. As soon as they started to come up I realized I had a new problem: uneven germination. Should I treat them in place or transplant them as they come up? Oryzalin (the active ingredient in Surflan) may prevent the ungerminated seeds from germinating if I just start spraying them in place. I decided to risk it and spray them in place.
Nothing much happened the first week. I sprayed them before I left for work/school every morning as opposed to in the evening, figuring that the chemicals would be more actively absorbed when the plant was more photosynthetically active. Week two was similar to the first. Week three I began to see plants starting to struggle, some cotyledons are looking pale. Week four I’m seeing plants withering and dying. For some pots I stopped spraying at week three, since I was seeing some high mortality. I definitely saw a lot of stunted growth. This took place over the months of March and April 2013. It’s now September 2013, and here are some of the results (pictured below).
Next time: Identifying Tetraploids.
Contreras, R.N. Interspecific Hybridization, Ploidy Manipulation, and Cytological and Genetic Analyses as Tools for Breeding and Improvement of Callicarpa L., Cryptomeria D. Don, Hibiscus L., and Tecoma Juss. Dissertation, University of Georgia. 2009