From qualitative to quantitative: a new approach for determining heat tolerance in cotton using triphenyl tetrazolium chloride
Background Susceptibility of cotton to heat stress in cotton production systems is a major concern for breeding programs. It is hypothesised that in order to maintain or improve cotton yields and quality in sub-optimal future climates, the negative effects of high temperature stress must be mitigated. To address this need, a fast and effect way of quantifying thermotolerant phenotypes is required. Triphenyl tetrazolium chloride (TTC) based enzyme viability testing following high temperature
... igh temperature stress can be used as a heat tolerance phenotype. This is because when live cells encounter a TTC solution, TTC undergoes a chemical reduction producing a visible, insoluble red product called triphenyl formazan, that can be quantified spectrophotometrically. However, existing TTC based cell viability assays cannot easily be deployed at the scale required in a crop improvement program. Results In this study, a heat stress assay (HSA) based on the use of TTC enzyme viability testing has been refined and improved for efficiency, reliability, and ease of use through four experiments. Sampling factors which may influence assay results such as leaf age, plant water status, and short-term cold storage were also investigated. Experiments conducted in this study have successfully down scaled the assay and identified an optimal sampling regime, enabling measurement of large segregating populations for application in breeding programs. The optimal durations of leaf disc exposure to TTC and the subsequent extraction of the formazan product in ethanol were identified as 16 h and 13 h, respectively; leading to enhanced clarity of assay results. Conclusions These improvements in the methodology provide a new level of confidence in results, ensuring applicability of the assay to a breeding program. The improved HSA methodology is important as it is proposed that long-term improvements in cotton thermotolerance can be achieved through concurrent selection of superior phenotypes based on the HSA and yield performance in hot environments. Additionally, a new way of interpreting both heat tolerance and heat resistance were developed to differentiate genotypes that perform well at the time of a heat stress event and those that maintain a similar level of performance to a non-stressed control.