The TTC-technique might not appropriately test the physiological stage of plant tissues

Abstract

The 2,3,5-triphenyltetrazolium chloride (TTC) technique has been used during decades to distinguish between dead and alive tissues of perennial grasses. This technique did not consider, however, that dormant (i.e., viable) tissues could exist within those erroneously considered dead tissues, thus being unable to report the true physiological stage of those plant tissues. Development of a procedure able to distinguish between metabolically active or dormant or dead tissues is then critical. This study developed a procedure to classify plant tissues of perennial grasses (Poa ligularis (Nees ex Steud.), Nassella longiglumis (Phil.) Barkworth, Amelichloa ambigua (Speg.) Arriaga & Barkworth, and Piptochaetium napostaense (Speg.) Hack.) in a more appropriate physiological stage (i.e., metabolically active, dormant or dead) than the traditional TTC-technique. Perennial grass seeds or meristematic buds were immersed in a TTC solution to obtain metabolically active (red or pink staining) or unstained (either dormant or dead) tissues. TTC-unstained tissues were placed in Evans Blue solution to separate dormant from dead tissues. The combination of TTC and Evans Blue techniques allowed the separation of metabolically active, dormant or dead tissues. Use of Evans Blue on TTC-unstained seeds and buds allowed to determine that some of these tissues were not dead but dormant (i.e., viable). Among the TTC-unstained tissues between 2 to 35% of total grass seeds and from 19.5 to 42% of all evaluated buds were dormant (viable and potentially able to growout) but not dead. Combination of TTC and Evans Blue techniques allowed a better classification of the physiological stages of plant tissues (metabolically active, dormant or dead) than the conventional TTC test.