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Hui-qing Li, Qing-he Li, Lei Xing, Gao-jie Sun and Xiu-lian Zhao

used in measuring cold hardiness ( Lindén et al., 2002 ). Among hardiness evaluation techniques, electrolyte leakage (EL) test and color reaction tests, such as TTC test, are widely used ( Palonen and Buszard, 1997 ). EL test is based on the principle

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Jean-Pierre Privé and M.I.N. Zhang

2,3,5-triphenyl tetrazolium chloride (TTC) staining, electrical conductivity, and electrical impedance (Z) analyses were used to assess freezing injury of `Beautiful Arcade' apple (Malus ×domestica Borkh.) roots taken in late March from either the field or 3C-refrigerated storage (cold-stored). Lethal temperature (LT50) levels using TTC or electrical conductivity occurred at colder temperatures than those found using Z. Techniques varied in their ability to detect changes in cell viability with increasing cold stress. Listed in order of decreasing responsiveness they are electrical impedance (Z), electrical conductivity, and TTC vital staining. With the most sensitive technique, Z, two parameters—extracellular and total tissue electrical resistance—were about five and eight times lower (indicating more injury) for roots from the field than from cold storage. The smaller values obtained from the field roots were probably due to natural in-field freeze-thaw cycling before the controlled cold-stress tests in the laboratory. More importantly, the impedance technique provided more detailed information than TTC or electrical conductivity about how apple roots respond to cold stress and how Z may provide some insight into freeze-thaw history before injury assessment. Although this technique shows potential, future studies are required to render a complete physiological significance to the impedance parameters involved in calculating Z.

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Weijie Jiang, Jie Bai, Xueyong Yang, Hongjun Yu and Yanpeng Liu

cryoprotective properties (such as proline and soluble sugars) and root TTC-reducing activity ( Ristic and Ashworth, 1993 ; Uemura et al., 1995 ; Xin and Browse, 2000 ). A number of genetic and molecular studies on plant response to low temperatures have

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Natalia R. Dolce, Ricardo D. Medina, Luis A. Mroginski and Hebe Y. Rey

. Seed viability from capsules of each treatment was determined using the TTC reduction assay ( Singh, 1999 ). Moreover, seed germination was determined by sowing seeds in 100-mL glass flasks containing 25 mL of solidified (0.65% agar A-1296; Sigma Chem

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Fumiomi Takeda, Rajeev Arora, Michael E. Wisniewski, Glen A. Davis and Michele R. Warmund

A seasonal study was conducted to assess the freezing injury of `Boskoop Giant' black currant (Ribes nigrum L.) samples from Oct. 1991 through Mar. 1992. Buds were subjected to either differential thermal analysis (DTA) or one of a series of temperatures (0 to -36C). Freeze injury was then assessed either visually or with TTC. Results indicated that black currant floral buds have multiple low-temperature exotherms (LTE). Freeze injury in intact buds could not be visually quantified because of the lack of visible browning, nor assayed with TTC reduction. Excised floral primordia incubated in TTC, however, developed colored formazan following exposure to nonfreezing and sublethal freezing temperatures, but remained colorless when exposed to lethal temperatures. The percentage of floral primordia that were colored and colorless were tabulated and a modified Spearman-Karber equation was used to calculate the temperature at which 50% of floral primordia were killed (T50 The T50 temperature was correlated with the temperature at which the lowest LTE was detected (R2 = 0.62). TTC reduction assay using excised floral bud primordia was a good indicator of viability in frozen blackcurrant buds. Chemical name used: 2,3,5-triphenyltetrazolium chloride (TTC).

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Mudau N. Fhatuwani and Makunga P. Nokwanda

recorded were soluble sugars (sucrose, glucose, and fructose), starch (amylopectin and amylase), total polyphenol content, TFC, total antioxidant activities (DPPH = 2,2-diphenyl-2-picrylhydrazyl and FRAP), and TTCs. The quality of bush tea from the

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Fumiomi Takeda, R. Arora, M. Wisniewski and M. Warmund

`Danka' black currant floral buds produce multiple low temperature exotherms (LTEs). However, the absence of visual injury symbtoms in the buds after exposure to subfreezing temperatures make it difficult to assess injury in these buds. A 2,3,5-triphenyltetrazolium chloride (TTC) reduction assay was used to determine whether LTEs corresponded to freezing injury of individual floral primordia or to the entire floral axis. Intact buds were cooled at 3C/n, removed at 3C intervals from -12 to -33C, and thawed on ice for 24 h. Duplicate samples were subjected to differential thermal analysis. Freeze injury Could not be measured with TTC in thawed, intact buds. However, incubation of excised floral primordia in TTC resulted in an all or nothing response. The number of LTES did not correspond to the number of floral primordia killed within a floral bud, but the median LTE did correspond with the temperature at which lethal injury of the whole inflorescence occurred. Therefore, preliminary results indicate that TTC reduction assay of individual floral buds is a fast, reliable technique to assess bud injury.

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Jean-Pierre Prive and M.I.N. Zhang

2,3,5-triphenyltetrazolium chloride (TTC) staining and electrical impedance (?) analyses of apple roots (Malus domestica Borkh. `Beautiful Arcade') taken in late March from either the field or from 3C refrigerated storage (cold-stored). LT50 levels using TTC were much lower than those found using electrical impedance. No loss of viability in the roots was detectable using TTC staining until a freeze–thaw stress of –9C whereas? analysis detected changes in cell viability after a freeze–thaw stress of only –3C. With increasing cold stress, two parameters: extracellular electrical resistance (Ro) and time constant?, decreased linearly for cold-stored roots and exponentially for field roots. Impedance analysis also revealed that the values for both extracellular Ro and total tissue electrical resistance (R?) for the field roots were approximately 5 and 8 times lower, respectively, than in the cold-stored roots. It is believed that the smaller Ro and R? values obtained from the field roots were due to natural in-field freeze–thaw cycling prior to the controlled stress tests in the laboratory. Based on the analyses of winter hardiness using the two methods, the impedance technique? provided the physiological information not only about the hardiness level, but also about freeze–thaw history prior to the hardiness assessment.

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Monte L. Nesbitt, Robert C. Ebel, Douglas Findley, Bryan Wilkins, Floyd Woods and David Himelrick

Containerized `Owari' satsuma mandarin (Citrus unshiu Marc.) on Poncirus trifoliata `Flying Dragon' rootstock were exposed to one of two acclimation regimes (cold acclimated and unacclimated) and frozen in a computer-controlled freezer to five different low temperatures. Whole plant survival was measured and compared to the results of four leaf and stem injury assays. Acclimating plants in growth chambers at 20 °C day and 10 °C night for 14 days, followed by 15 °C day and 4 °C night for 14 to 21 days resulted in an 81% and 80% increase in leaf and stem survival, respectively, when frozen to a low of -8 °C. Electrolyte leakage and phenolic leakage assays effectively detected changes in percent leaf survival, but the TTC stain assay, using leaf disks, did not. Stem survival was best predicted by the TTC assay, using the phloem as the indicator tissue for survival. Electrolyte leakage and phenolic leakage were also reliable assays for predicting stem survival, although survival percentages were different at the same electrolyte leakage values reported in other studies. The callus growth assay accurately predicted survival for cold acclimated satsuma mandarin stems only. Chemical name used: triphenyl tetrazolium chloride (TTC).

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D.O. Ketchie and R. Kammereck

Differential thermal analysis (DTA) and tetrazolium triphenyl chloride (TTC) were done on shoots of 4-year-old `Braeburn' apple trees for 3 years. The trees acclimated slowly in autumn. If cold temperatures last long enough in winter, shoots will acclimate as low as –40C. Shoots are sensitive to warm temperatures and deacclimated rapidly. An attempt to run a controlled test on freeze resistance of `Braeburn' did not respond to DTA. Moisture samples indicated trees were freeze dried. Different sets of trees were rehydrated and showed an exotherm pattern. Exotherms could be seen after 3 days at 26C, 14 days at 10C, and 21 days at 4C. Another controlled freeze test was performed on 1-year-old `Braeburn' trees. Trees were acclimated outdoors. An exotherm pattern could be seen upon DTA analysis. After artificial freezing, DTA and TTC tests showed pith killed at –24C, primarily xylem at –28C, and all tissue at –35C. After freezing, trees were placed in a greenhouse and warmed over 2 months. Upon dissection, we found xylem produced before freezing was dead, but a large amount of new xylem was generated. Trees appeared to have normal leaf and shoot growth for about a month, but eventually wilted and died. Dissection of these showed the same results as the first set dissected. New xylem evidently was not enough to carry the growth of the trees.