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 distin guish 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 metabol ically 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 physiologi cal 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 sepa rate 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 poten tially able to growout) but not dead. Combination of TTC and Evans Blue techniques allowed a better classifi cation of the physiological stages of plant tissues (metabolically active, dormant or dead) than the conventional TTC test.

Research on the reproductive biological characteristics of Amomum villosum Lour. and Amomum longiligulare T. L. Wu

“…longiligulare ’ pollen also decreased to 0.00% (in vitro germination) and 7.66% (TTC staining method), the fruit setting rate was still high for pollen stored for 36 h ( Fig 5B ). Although the TTC staining method has the problem of false positives in other experiments [ 22 ], in this study, the TTC staining method can detect pollen viability more reliably and is simpler. The basic trend reflected by the in vitro germination method was similar to the TTC method; however the in vitro germination method was often restricted for various reasons, resulting in a low germination rate that was unable to reflect the true pollen vitality [ 23 ].…”

How different reproduction protocols can affect the germination of seeds: The case of three stenoendemic species on Mt. Olympus (NC Greece)

“…The method used for staining was: -Staining with a 1% w/v tetrazolium chloride solution (abbr. TTZ) (ISTA 1999), and -Staining with 0.25% w/v Evans blue solution (Busso et al 2005;Busso et al 2015) The embryos were treated with tetrazolium chloride solution, and the embryos that were not stained and therefore initially considered to be non-viable were then immersed in a solution of Evans blue dye. Thus, the percentage of non-viable embryos is the percentage of stained embryos after the Evans blue test.…”

“…Both solutions were used due to the different properties of their active substances. Tetrazolium chloride solution only stains tissues red that are metabolically active (so-called viable tissues), whereas the Evans blue solution only stains dead tissues, which results in an accurate evaluation of embryo viability (Busso et al 2015).…”