Optimized qualitative and quantitative methods for barley viability testing using triphenyl tetrazolium chloride staining

Abstract

BACKGROUND AND OBJECTIVES: Barley viability is an important agronomic trait and has a direct influence on subsequent beer production. Traditional triphenyl tetrazolium chloride (TTC) test evaluating seed viability still has some drawbacks, for example, excessive instability and time consumption. In the present study, we optimized both the qualitative and quantitative TTC seed viability test methods, and established more accurate and convenient assays to evaluate barley seed viability. FINDINGS: In the qualitative evaluation method, we established the vigor index according to the proportion of staining embryos, allowing quick evaluation of both germination and mortality rates in <12 hr. In the quantitative evaluation method, we optimized TTC incubation time, extraction temperature, and solvent volume to tissue ratios making results more reliable. Finally, we developed a high‐throughput method by combining freeze drying and tissue lysing instead of the time‐consuming method of grinding on liquid nitrogen. There was a positive significant statistical correlation between germination rate and formazan content by the optimized quantitative evaluation method (R² = 0.907; p < 0.01). CONCLUSIONS: Both the optimized qualitative and quantitative TTC seed viability test methods allow a quick and reliable seed vigor evaluation for different varieties or different aged barley seeds. SIGNIFICANCE AND NOVELTY: The optimized seed vigor evaluation method can be used to effectively screen out low vigor barley seeds. The qualitative evaluation method is appropriate to quickly evaluation for a small number of samples in <12 hr. The high‐throughput quantitative evaluation method is fit for a large number of sample evaluations as much as 80 samples at the same time.

Plant phenotyping relevance

大麦種子の生存性・活力という植物状態を測定するTTC法を最適化し、定量法を検証して高スループット化しているため、表現型取得法が研究の中心です。

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