A standardised approach for determining heat tolerance in cotton using triphenyl tetrazolium chloride

Improving the heat tolerance of cotton is a major concern for breeding programs. 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 stress can be used as a vegetative 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. 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 that 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 downscaled the assay and identified an optimal sampling regime, enabling measurement of large segregating populations for application in breeding programs. The improved HSA methodology is important as it is proposed that long-term improvements in cotton thermotolerance can be achieved through the 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 was developed, differentiating genotypes that perform well at the time of a heat stress event and those that maintain a similar performance level to a non-stressed control.

Biometric characterization and tetrazolium test in Campomanesia phaea O. Berg. Landrum seeds

ABSTRACT: Campomanesia phaea has commercial interest because its fruits are used in the manufacture of several products, besides its high nutritional potential. Nevertheless, it has low germination potential and there is not much information in the literature. The aim of this study was to perform a biometric characterization and to establish the tetrazolium test for cambuci seeds. Seeds obtained from two collection periods were subjected to biometric evaluations (seed length, thickness, width and mass), water content, determination of the soaking curve and tetrazolium test. The soaking curve was performed with non-scarified seeds and seeds subjected to two scarification methods, mechanical and chemical. The tetrazolium test was performed soaking the seeds in a solution of 2, 3, 5 triphenyl tetrazolium chloride salt at 0.75, 1.00 and 1.50% for 4, 8 and 12 hours. After this period, the seeds were categorized as viable and non-viable. It was concluded that the cambuci seeds presented biometric differences between the two collection periods, because of the different water contents. There was no difference in the soaking curve among the three treatments tested and the solution of 0.75% for four hours in a water bath at 38 oC was considered the best for performing the tetrazolium test.

Seed quality testing study in Quinoa (Chenopodium quinoa Wild.)

Present study was conducted on four genotypes of quinoa to find out optimum temperature and method for germination test at Department of Seed Science and Technology, CCS HAU, Hisar during 2019-20. The result of this study revealed that at 25oC temperature average germination was 22.47 per cent while it was 13.31 per cent at 20oC temperature. Similarly seedling lengths were 9.69 and 7.58cm and seedling vigour index-I were 229.54 and 103.07 at 20 and 25oC respectively. Between the methods, Top of Paper (TP) recorded 19.78 % germination while Between Paper (BP) recorded 16.00 % germination. Similarly, 8.51 and 8.77cm seedling lengths and 157.83 and 174.78 vigour index-I were observed in Top of Paper and Between Paper methods respectively. Poor germination was recorded in all four genotypes (Maximum 42%) which was confirmed by conducting tetrazolium test. It is concluded from the study that 25 oC temperature and both Top of Paper and Between Paper methods can be used for germination test in quinoa crop. For radical emergence test, 250C temperature for 60 hours should be used and for seed viability test, the seeds should soaked into 1.0% percent tetrazolium solution (2, 3, 5-triphenyl tetrazolium chloride) for 20 hours at 30oC.

Evaluation of freezing injury in temperate fruit trees

Freezing is a major environmental stress limiting the geographical distribution, growth, and productivity of temperate fruit trees. The extent of freezing injury in the trees depends on the rate at which the temperature decreases, the minimum temperature reached, and the duration of the freezing conditions. The ability to tolerate freezing temperatures under natural conditions varies greatly among fruit tree species, cultivars, and tissues. Freezing injury must be precisely evaluated to reliably predict the winter survival and productivity of the trees in specific regions, to screen for tolerant species and cultivars, and to develop cultural strategies that reduce freezing stress. Various methods are used to evaluate freezing injury in temperate fruit trees under field and artificial conditions, including visual evaluation of tissue discoloration, thermal analysis, determination of electrolyte leakage, and triphenyl tetrazolium chloride reduction analysis. In this review, we describe the most frequently used experimental procedures for evaluating freezing injury.

The Submergence of the Graft Union Causes the Death of Grafted Mango Trees (Mangifera indica L.) under Flooding

The risk of flooding has increased as a result of climate change inspired by global warming. In this study, the difference in tolerance to flooding between grafted trees and seedlings was investigated in mango trees (Mangifera indica L.). Two levels of flooding treatment were established, in addition to a non-treated control. One was “30-flood,” in which trees were submerged up to 30 cm from the soil surface, and another was “10-flood,” in which trees were submerged up to 10 cm from the soil surface. Grafted trees treated with the 30-flood were submerged up to 5 cm above the graft union. Only the grafted trees in the 30-flood treatment began to decrease Fv/Fm and the root sugar content during treatment, then died after the completion of treatment. In grafted trees and seedlings alike, both flooding treatments also reduced root vigor, as determined by triphenyl tetrazolium chloride reduction tests and root starch content. However, root vigor and root starch content in grafted trees treated with the 10-flood as well as seedlings treated with both 10- and 30-flood recovered within two months after the completion of treatment, so that no significant differences were detected between the control. These results suggest that the submergence of the graft union into water causes the decline in photosynthesis of the scion first and the death of the grafted trees as a result.

Potential Fertilization Capacity of Two Grapevine Varieties: Effects on Agricultural Production in Designation of Origin Areas in the Northwestern Iberian Peninsula

In the present study, we analyzed the main parameters related with the potential fertilization ability of two grapevine varieties, Godello and Mencía, during the years 2017 and 2018. The research was carried out in two vineyards of the Galician winegrowing Designation of Origin areas of Ribeiro and Ribeira Sacra. Ten vines of each variety were selected for bunch and flower counting, pollen calculations, pollen viability studies by means of aceto-carmine (AC) stain and 2, 3, 5-triphenyl tetrazolium chloride (TTC) methods, and the determination of their germination rate. In all vineyards the 50% fruitset was reached, except for Godello in Cenlle during 2017. The mean coulure value was higher for Godello (40.5%) than for Mencía (31%). Analyzing the pollen production per plant and airborne pollen levels, we observed important discordances between them, which can be due to the influence of weather conditions and be related with self-pollination processes. We found important differences on pollen viability depending on the applied method and variety, with higher values for the AC method than the TTC for both varieties in all study plots, and higher values for Mencía variety than Godello. Regarding germination rates, we observed a marked reduction in 2017 with respect to 2018, in all study sites and for both varieties. The analyzed parameters were useful to explain the different productive abilities of Godello and Mencía varieties in the two studied bioclimatic regions of Ribeiro and Ribeira Sacra.

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 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.