Hello everybody! I am analyzing the impact of temperature-induced aging on the viability of various Arabidopsis accessions. To do this, I incubate the seeds in a solution of 1% 2,3,5 triphenyl tetrazolium chloride for 72 hours at 30 ° C in the dark. For some accessions, such as Col-0 (Columbia), it is fairly easy to distinguish between viable and non-viable seeds, and few seeds have intermediate staining. However, other accessions such as Ws (Wassilewskija), show high heterogeneity with many partially stained seeds, even for controls, and it becomes very difficult to distinguish between viable and non-viable seeds. This has already been described in some publications since Ws seed coat seems to be slightly permeable for tetrazolium.
I have tried to improve this staining by washing with a scarification solution (20 ml commercial bleach and 100 µl Triton X-100 in 100 ml autoclaved distilled water), or increasing incubation time, but was unsuccessful. Any recommendations to improve this assay? Thank you very much for reading me.
Hello! Nacho Lescano Sir, here I have tried to answer your problem as per my knowledge..
Sometimes there is partial staining in the Tetrazolium Test but if it seems that there is enough staining at critical parts of the seed then that seed is considered viable, although such seed lots usually have low vigour. Those seeds may be old or may be damaged by heat/ moisture and insects therefore will have low viability.
Here you have not mentioned that seeds which you used for incubation in 1% 2,3,5 triphenyl tetrazolium chloride were presoaked in distilled water for overnight or not? If not then please try to presoak the seeds in distilled water for overnight after that incubate with the tetrazolium solution. This may help you.
Thank's for your kind response Vagmi Singh ! As you recommended, I stained the seeds after ON incubation in distilled water. However, the result was the same. These seeds present 100% germination after 24 hours of light exposition under short photoperiod. TZ test is used also as a coat permeability assay and seems that Ws accesion present low permeability compared to others (like Col-0). Article Influence of the Testa on Seed Dormancy, Germination, and Lo...
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