Tips on doing the Triple Response assay (and other ethylene/ACC–related assays) right
(by Anna Stepanova)
The Triple Response Assay and Its Use to Characterize Ethylene Mutants in Arabidopsis. Merchante C, Stepanova AN. Methods Mol Biol. 1573:163-209. doi: 10.1007/978-1-4939-6854-1_13. (2017)
The media we use is AT [1x MS salts, 1% sucrose, pH 6.0 w/1M KOH, 0.6-0.8% bacto-agar; autoclave for 20-30 minutes]. 10ppm ethylene or 10uM ACC (added after autoclaving) can be used for the triple response assay. The ethylene insensitivity of classical ethylene mutants like that of ein2 and ein3 is very obvious in these conditions: the mutants literally stand out when grown next to wild-type plants (See picture below). For visualizing the phenotypes of very weak ethylene mutants, we often use either 0.2 or 0.5uM ACC. For otherwise incompletely penetrant mutants (like our wei8) lower ACC seems to do the trick and gives a more or less uniform ethylene insensitivity compared to wild type.
We sterilize seeds with 50% bleach (spiked with Triton at 2-5 drops per 0.5L, which makes the seeds more “wettable” and allows the clumps to break) for 5-15 minutes, wash with sterile water 3 or more times, resuspend the seeds in melted pre-cooled 0.7% low-melting-point agarose in water, and plate by spreading the seeds [using a 200uL pipetman with a sterile wide-bore tip] on the surface of an AT plate supplemented with ACC. To have roots enter agar nicely (as opposed to them growing on the surface of the plate), it helps to have the plates with media air-dry for 2-3 days at room temperature at a bench (keep the lids closed) prior to seed planting. Also, the volume of top-agarose seems to matter (we typically use 80-100ul of top agarose per up to 100 seeds, and spread the entire volume per 1/10 sector or a larger area of a standard 100mm Petri dish). I like plating the seeds in straight lines, so it is easier to see the roots of weaker mutants. It helps to have the controls planted side by side (we routinely plate ~10 lines per Petri dish).
Once planted, keep the plate(s) at 4ºC for 2-5 days (typically, 3) to equalize germination (no need to wrap or tape the plates). After the cold treatment, we usually light-treat the plates with seeds for about 2 hours at room temperature (that seems to improve germination), and then cover the plate(s) with aluminum foil and place them horizontally in the 22ºC dark incubator (stacking plates is OK, just do not mix plus and minus ACC plates together in the same stack). After about 72 hours, we unwrap the plates and look at them. The roots are easier to see from the bottom side of the plate. For quantification purposes, we pull the seedlings out of agar and place them horizontally side-by-side on the surface of another AT plate, scan the images and quantify organ length using NIH Image or another program.
Other thoughts: spread the seeds nicely, do not overdo in terms of density (no more than 50 seeds per 1/10 sector of a Petri dish, preferably less). I personally do not like using vertical plates with ACC (OK for ethylene), as not every root touches the plate equally, resulting in a lot of variability between seedlings (the less contact with the ACC-containing media, the longer the root). For quantification, I always use very fresh seeds and typically propagate my controls side-by-side with the mutants (i.e. preferably, in the same tray of soil). If using very fresh seeds, make sure they are fully dry (1-2 days at room temperature post harvesting is usually enough). And I don’t like the idea of thinly poured plates (25-30 mL media per 100mm plate is what we typically use), as then you have to worry about uneven plate drying, different top-agarose volumes, proper leveling, and other undesirable variables. Too much media in plates is also not good: the taller ethylene insensitive seedlings end up touching the lids and getting wet.
And the final note: don’t forget to include your controls: a wild-type and an ein, especially when trying the assay for the first time. Good luck!