Identification of cold resistance in Vitis amurensis germplasms base on TTC staining index and Logistic equation

Abstract: Amur grape (Vitis amurensis Rupr.) is the most cold-resistant species in Vitis, and the important parent material in hardiness breeding. For the cold resistance, the dormant shoots have high heritability. However, there might be some differences on cold hardiness among amur grape germplasms in the field. A prerequisite to conducting research in the area of cold hardiness is a reliable method to determine tissue viability. So far, there were no readily available methods to evaluate cold resistance of amur grape shoot, which affected the further excavation and utilization of cold-resistant germplasm. Primarily the triphenyl tetrazolium chloride (TTC) test was used in the identification of cold hardiness for many woody plants as a qualitative response, and if the red formazan derivative was produced, the tissue was considered to be viable, but whether this convenient method was suitable for amur grape or how to use it was not clear. The research was to set up a quick and reliable method for cold-resistant evaluation in V. amurensis germplasms at the dormancy stage. The 40 one-year-old dormant shoots of amur grape germplasms were collected as test materials. Eight freezing temperatures were ?15, ?20, ?25, ?30, ?35, ?40, ?45 and ?50 ℃, respectively. The chill treatment lasted 12 h and then the germplasms were recovered for 4 h at room temperature after chilling treatment. Both the cooling and heating rates were set 4.0 ℃/h. Then 10 sections with the thickness of 5 cm were cut from the shoots, added with 10 mL 0.5% TTC and dyed for 4 d in 30 ℃ incubator avoiding light. The staining areas of the longitudinal section were projected onto an LA2400 scanner attached to a WinRHIZOTM Image Analysis System (Regent Instruments Inc., Canada). The level of cold injury of each shoot was recorded as 5 levels based on the area of dyeing in the whole longitudinal section. Then staining index was calculated at related level. The results showed that staining indices and related freezing temperatures presented a typical inverted S-shape curve, moreover, the lower the treatment temperature, the smaller the dyeing index of the shoots, so the staining indices could reflect the changing process of shoot cold injury after freezing treatment. Therefore, the cold resistance of each germplasm was determined from the low semi-lethal temperature values using a logistic equation of staining indices, the low semi-lethal temperature was from ?39.41 to ?29.75 ℃, and the fitting degree was from 0.956 4 to 0.999 3 for 40 V. amurensis germplasms based on pictorial assessment of TTC staining image. The correlated analysis of low semi-lethal temperature values by fitting staining indices with average subordination values by subordinate function analysis and field germination rates were highly significantly negative correlation. The correlation coefficient was ?0.883 03 and ?0.876 70 respectively, which could reflect the actual activity of cold hardiness in the field. In conclusion, the method of pictorial assessment of TTC staining image is quick and convenient to operate, which has a certain feasibility and accuracy on the cold-resistant evaluation of V. amurensis germplasms at dormancy stage.