Abstract
Cold hardiness evaluation is important for screening woody species in cold areas. We compared cold hardiness by estimating the 50% lethal temperature (LT50) using electrolyte leakage test (ELLT50) and triphenyltetrazolium chloride test (TTCLT50) for 26 woody species in the Bashang region of China. One-year-old shoots were collected in January and exposed to five subfreezing temperatures in a programmable temperature and humidity chamber. LT50 was estimated by fitting relative electrolyte leakage and percentage of dead tissue against test temperature. For all tested species, triphenyltetrazolium chloride (TTC) staining of the pith was weak and the cambium TTCLT50 was lower than the extreme minimum temperature (−37 °C) recorded in the region. The cambium TTCLT50 and the sd were lower than that for the phloem and xylem. The phloem TTCLT50 was lower than the xylem TTCLT50, and the two sds were similar. The ELLT50 showed no significant correlation with any TTCLT50. For most species, the ELLT50 was higher than the cambium and phloem TTCLT50 and was not significant different with the xylem TTCLT50. The ELLT50 showed higher sd than any tissue TTCLT50. Based on results obtained in this study, when choosing cold hardiness of single stem tissue as an indicator for screening woody species, the xylem should be considered first, followed by the phloem; the cambium and pith were unsuitable. The cold hardiness estimated by ELLT50 was more suitable as indicator for screening woody species than that of stem tissue in winter estimated by TTCLT50.
Cold hardiness is the ability of a plant or plant organ to tolerate freezing or survive freezing conditions (Fuchigami, 1996) without sustaining injury (Lindén et al., 2002; Weiser, 1970), which is a major determinant of plant species growth and distribution. Cold hardiness fluctuates between the seasons. During winter, plant species show increased tolerance to low temperatures with cold acclimation. The winter cold hardiness of a species is an important factor that ultimately affects species survival.
Cold hardiness evaluation is crucial for screening woody species in cold areas. Many methods have been used to quantify injury to woody stem pieces following laboratory freezing (Calkins and Swanson, 1990). The LT50 is most commonly 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 that damage to cell membranes results in enhanced leakage of electrolytes (mainly K+) from the cell (Lindén, 2002; Pukacki and Pukacka, 1987) and increases electrical conductivity (EC). LT50 estimated using the EL test (ELLT50) means the temperature results in 50% increase in EL. Cell membranes are believed to be the primary sites of injury caused by stressful environmental conditions such as freezing temperatures (Lyons et al., 1979). EL test is an indirect measurement of the integrity of cell membranes (Steffen et al., 1989). Relative EL (initial EC1/final EC2) estimates the degree of cell membrane injury (Nesbitt et al., 2002), where EC1 is EC after freezing exposure and EC2 is EC after total disruption of cell membranes. A disadvantage of the EL test is that leakage among different tissues is not distinguished (Calkins and Swanson, 1990). The TTC test is based on the principle that living plant tissue can reduce TTC in the mitochondria to produce red formazan, whereas dead tissue cannot (Nesbitt et al., 2002). LT50 estimated with TTC test (TTCLT50) means the temperature that results in 50% decrease in TTC reduction, 50% red formazan, and 50% no staining. TTC reduction reflects the activities of some dehydrogenases in the respiratory transport system (Sakai and Larcher, 1987). It measures the freezing injuries to metabolic activity (Lassheikki et al., 1991). The primary advantage of the latter method is that death in specific tissue can be detected, but visual classification of the staining intensity may be difficult (Purcell and Young, 1963) and is subjective (Nesbitt et al., 2002). According to Sakai (1955), freezing injury of twigs first appears as a brown ring at the peripheral layers of the xylem and pith tissue. Cold hardiness estimation of stem using the TTC test has been mainly focused on the phloem (Sharma and Graves, 2004; Soloklui et al., 2012) and xylem (Zhang et al., 2012). Although time-consuming, tissue browning is still generally used due to its reliability and ability to predict (Calkins and Swanson, 1990). In the present study, we use the M percentage in the CMYK color system (C: cyan; M: magenta; Y: yellow; K: black) using Adobe Photoshop (CS2) software (Adobe, San Jose, CA) to estimate objectively and quantitatively the staining intensity of stem pieces. This approach overcomes the afore-mentioned disadvantages and enables staining assessment more easily and precisely.
No single method is reliable for all materials (Calkins and Swanson, 1990). The objectives of this study were 1) to compare cold hardiness of the cambium, phloem, xylem, and pith for woody species by TTC staining and TTCLT50; and 2) to compare cold hardiness estimations of woody species by ELLT50 and TTCLT50.
Materials and Methods
Study area.
The Bashang region of China is a transitional zone between the North China Plain and Inner Mongolian Plateau, and is a typical agricultural-pastoral plateau ecotone. The vegetation is dominated by semiarid grassland. Woody plants play an important environment protection function. The southern part of the region is an agricultural area and the northern part comprises grassland. Elevation ranges from 1000 to 1700 m and is typically higher (1400–1700 m) in the north. The winter is cold with the average monthly temperature is below 0 °C for 5 months. The 30-year extreme minimum temperature in the region is −37 °C, the minimum temperature in January (the coldest month) in most years is −30 °C, and the 30-year mean annual minimum temperature is −22 °C. The average wind speed is 4.9 m·s−1 for the entire year and the highest wind speed recorded is 27 m·s−1. Low temperature poses a severe challenge to woody species survival in winter.
Plant material.
Woody plant species were sampled from the Ping Ding Bao forestry farm (lat. 41°40′N, long. 115°40′E, 1410 m) and the Tun Ken forestry farm (lat. 41°54′N, long. 114°47′E, 1450 m) in the Bashang region of China. In total, 26 species belonging to 11 families were sampled, comprising 5 evergreen conifer species and 21 deciduous broadleaf species (Table 1). Using individual plant parts to evaluate cold hardiness is the usual method in cold hardiness tests (Hoffman et al., 2010; Lisek, 2012). Thirty 1-year-old shoots (30–40 cm long) from the upper portion of the juvenile stage of each species were randomly collected in January. Most of the plants sampled were 6 to 7 years old. The cut wound was sealed with paraffin, and then the sampled shoots were stored in plastic bags at 0 °C in a cold room until the freezing test (all freezing tests were finished in less than 1 month).
Cold hardiness evaluation of 26 woody species by estimation of ELLT50 and TTCLT50.
Freezing procedure.
The sampled shoots were transferred to a laboratory from the 0 °C cold room. For each species, the samples were divided into six sets: five sets were used for freezing tests and one set as nonfrozen control. Each set had five shoots.
Ice nucleation is considered crucial to ensure the accuracy of freezing tests (Workmaster et al., 1999). Each shoot for the freezing tests was rinsed with water, wrapped in moist cheese cloth and enclosed in aluminum foil (Ndlovu, 2015), placed in plastic bags, and transferred to a programmable temperature and humidity chamber (UK-80G; Qin Zhuo Environmental Equipment Company, Dongguan, China). The initial temperature was −2 °C. Materials were kept at −2 °C for 24 h to equilibrate and ensure freezing. The cooling rate was −2 °C·h−1. This rate provides adequate time for intracellular water to migrate to freezing sites (Bigras and Colombo, 2001). The test temperature (−15, −25, −35, −45, and −55 °C) was maintained for 6 h. The warming rate was 2 °C·h−1 and the final temperature was 4 °C. After thawing for 12 h at the final temperature, the shoots were transferred to room temperature in a black bag for ≈12 h until the materials were evaluated.
EL test.
Shoots were rinsed with deionized water and dried with filter paper; 0.2- to 0.5-cm-long sections were cut from the apical, middle, and basal parts of each shoot. One gram was weighed. The number of replicates was five. Samples were immersed in 15 mL deionized water in a tube, incubated at room temperature (22 °C) for 11 h, then shaken for 1 h at 200 rpm on a THZ-22 constant temperature oscillator (Pei Ying Experiment Equipment Corporation Ltd, Taicang, China), and kept at room temperature (22 °C) for 12 h. EC1 was measured using a DDSJ-30A conductivity meter (Shanghai Precision and Scientific Instrument Corporation Ltd, Shanghai, China). Then the samples were killed in a boiling water bath for 20 min, kept at room temperature (22 °C) for 11 h, shaken for 1 h, and then kept at room temperature (22 °C) for 12 h, and finally EC2 was measured. Relative EL (Rt) was calculated as Rt (%) = (EC1/EC2) × 100.
TTC test.
One 1-cm-long section was cut from the apical, middle, and basal parts of each shoot, and placed in a tube, to which 10 mL 0.5% 2,3,5-triphenyltetrazolium chloride (TTC) was added. The number of replicates was five. Samples were incubated for 24 h in the dark at room temperature and photographed using a light microscope (Bx53; Olympus, Tokyo, Japan). Stained tissues were coded as living and nonstained tissues as dead. Staining intensity of the cambium, phloem, xylem, and pith was separately estimated by the photographs using Adobe Photoshop (CS2) software (Adobe Photoshop-Window-Info-M). M (magenta) percentage in the CMYK color system indicates the percentage of living tissue.
Estimation of LT50..
Grading of species.
Based on the LT50 and the minimum temperature in January (the coldest month) in the Bashang region, we classified the sampled species into four grades: Grade I species, LT50 ≤ −37 °C (lower than the extreme minimum temperature in the region); Grade II species, −37 °C < LT50 ≤ −30 °C (between the extreme minimum temperature in the region and the minimum temperature recorded in most years); Grade III species, −30 °C < LT50 ≤ −22 °C (between the minimum temperature recorded in most years and the 30-year mean annual minimum temperature); and Grade IV species, −22 °C < LT50 (higher than the 30-year mean annual minimum temperature).
Statistical analysis.
Mann-Whitney test, descriptive statistics, and Spearman correlation analysis were performed using OriginPro 9.1 software.
Results
Estimation of cold hardiness by TTC staining and TTCLT50
Pith cold hardiness.
For individual species, TTC staining intensity of stem was ranked as cambium > phloem > xylem > pith (Fig. 1A–C). TTC staining of the pith was weak. The staining intensity of Swida alba exposed to −55 °C was close to zero (Fig. 1B), thus no LT50 estimate was calculated for the pith.
TTC staining intensity estimation using M% in the CMYK color system by Adobe Photoshop software on the stem photographs of 1-year-old shoots of woody species exposed to subfreezing temperatures. (A) Poplar 84K exposed to −55 °C. Phloem: 67%; Cambium: 70%; Xylem: 51%; Pith: 7%. (B) Swida alba exposed to −55 °C. Phloem: 61%; Cambium: 77%; Xylem: 51%; Pith: 4%. (C) Populus simonii exposed to −15 °C. Phloem: 68%; Cambium: 74%; Xylem: 49%; Pith: 10%. TTC = triphenyltetrazolium chloride. M% = magenta percentage, which indicates the percentage of living tissue; CMYK = C: cyan; M: magenta; Y: yellow; K: black.
Citation: HortScience horts 55, 8; 10.21273/HORTSCI14930-20
TTC staining intensity estimation using M% in the CMYK color system by Adobe Photoshop software on the stem photographs of 1-year-old shoots of woody species exposed to subfreezing temperatures. (A) Poplar 84K exposed to −55 °C. Phloem: 67%; Cambium: 70%; Xylem: 51%; Pith: 7%. (B) Swida alba exposed to −55 °C. Phloem: 61%; Cambium: 77%; Xylem: 51%; Pith: 4%. (C) Populus simonii exposed to −15 °C. Phloem: 68%; Cambium: 74%; Xylem: 49%; Pith: 10%. TTC = triphenyltetrazolium chloride. M% = magenta percentage, which indicates the percentage of living tissue; CMYK = C: cyan; M: magenta; Y: yellow; K: black.
Citation: HortScience horts 55, 8; 10.21273/HORTSCI14930-20
TTC staining intensity estimation using M% in the CMYK color system by Adobe Photoshop software on the stem photographs of 1-year-old shoots of woody species exposed to subfreezing temperatures. (A) Poplar 84K exposed to −55 °C. Phloem: 67%; Cambium: 70%; Xylem: 51%; Pith: 7%. (B) Swida alba exposed to −55 °C. Phloem: 61%; Cambium: 77%; Xylem: 51%; Pith: 4%. (C) Populus simonii exposed to −15 °C. Phloem: 68%; Cambium: 74%; Xylem: 49%; Pith: 10%. TTC = triphenyltetrazolium chloride. M% = magenta percentage, which indicates the percentage of living tissue; CMYK = C: cyan; M: magenta; Y: yellow; K: black.
Citation: HortScience horts 55, 8; 10.21273/HORTSCI14930-20
Cambium cold hardiness.
Mann-Whitney test showed the cambium TTCLT50 was significantly different from the phloem and xylem TTCLT50 (Table 2). The cambium TTCLT50 was lower than the phloem and xylem TTCLT50 (Table 1), thus the cambium showed higher cold hardiness than the phloem and xylem. The cambium TTCLT50 showed a higher correlation with the phloem TTCLT50 than that shown by the xylem TTCLT50 (Table 3). The cambium TTCLT50 showed a lower sd than the phloem and xylem TTCLT50 (Table 4). For all tested species, the cambium TTCLT50 was lower than the extreme minimum temperature in the region (−37 °C). On the basis of the cambium TTCLT50, all tested species were classified in Grade I. Pinus sylvestris could tolerate −68 °C, whereas Caragana korshinskii could tolerate −37 °C.
Mann-Whitney test for the differences of special 50% lethal temperature (°C) (LT50) for wood stem tissues.
Spearman correlation coefficients between ELLT50 and TTCLT50 for wood stem tissues.
Descriptive statistics for ELLT50 and TTCLT50.
Phloem cold hardiness.
Mann-Whitney test showed the phloem TTCLT50 was significantly different from the xylem TTCLT50 (Table 2). The phloem TTCLT50 was lower than that for the xylem (Table 1); therefore, the phloem cold hardiness was higher than of the xylem. The phloem TTCLT50 showed a similar sd with the xylem TTCLT50 (Table 4). Based on the phloem TTCLT50, two species were classified in Grade II and two species belonged to Grade III, and for 22 species the phloem TTCLT50 was lower than the extreme minimum temperature (−37 °C). Pinus sylvestris could tolerate −57 °C, whereas C. korshinskii could tolerate −25 °C. Grade I species comprised Pinus sylvestris, Rosa davurica, Larix gmelinii, Populus alba, Populus simonii, Salix limprichtii, Populus alba × Populus glandulosa, Picea asperata, Populus alba var. pyramidalis, Armeniaca sibirica, Prunus cerasifera, Betula platyphylla, Ulmus pumila, Rubus corchorifolius, Swida alba, Populus gansuensis, Juniperus rigida, Hippophae rhamnoides, Amygdalus triloba, Populus ×beijingensis, Populus lasiocarpa, and Syringa oblata.
Xylem cold hardiness.
All studied species showed lower cold hardiness of the xylem, compared with the phloem (Table 1). P. sylvestris xylem tolerated −25 °C, whereas the phloem tolerated −57 °C. Based on the xylem cold hardiness, 7 species belonged to Grade I, 7 species were classified in Grade II, 10 species belonged to Grade III, and 2 species were categorized as Grade IV. Grade I species consisted of P. alba, R. corchorifolius, P. simonii, P. alba var. pyramidalis, Populus ×beijingensis, B. platyphylla, and S. alba, of which four species belonged to the genus Populus.
Cold hardiness estimation by TTC and TTCLT50 for 26 woody species revealed that the cambium showed the highest cold hardiness (Table 1) and the lowest sd (Table 4). The phloem showed higher cold hardiness than the xylem and similar sd with the xylem (Table 4).
Estimation of cold hardiness by ELLT50
Mann-Whitney test on the studied species showed the ELLT50 was significantly different from the cambium and phloem TTCLT50 (Table 2). The ELLT50 also showed no significant correlation with any TTCLT50, including the cambium TTCLT50, the phloem TTCLT50, and the xylem TTCLT50 (Table 3). That meant the relationship between the ELLT50 and TTCLT50 varied with species. For most species, the ELLT50 was higher than the cambium and phloem TTCLT50 (Table 1). That meant the species cold hardiness estimated by ELLT50 was generally lower than the cold hardiness of cambium and phloem estimated by TTCLT50. Descriptive statistics showed mean value of the ELLT50 was close to that of the xylem TTCLT50 (Table 4). Mann-Whitney test showed there was no significant difference between the ELLT50 and the xylem TTCLT50 (Table 2), but for some individual species like Pinus sylvestris, there was still a big difference between the LT50 values estimated by these two methods. That meant the cold hardiness estimated by ELLT50 was generally not significantly different from that of the xylem estimated by TTCLT50.
The cold hardiness of the test species estimated by ELLT50 varied significantly (Table 1) and showed the highest sd (Table 4). P. sylvestris tolerated −64 °C, whereas J. rigida was tolerant of only −16 °C. P. sylvestris, P. asperata, A. sibirica, P. gansuensis, C. korshinskii, R. corchorifolius, and R. davurica were tolerant of much lower temperatures than the extreme minimum temperature (−37 °C) in the region. In particular, P. sylvestris and P. asperata were tolerant of temperatures lower than −50 °C.
Discussion
Evaluation of cold hardiness estimated by TTCLT50.
Standard deviations (Table 4) showed the variation of cold hardiness among different species. Low sd means small variation of cold hardiness among different species. Indicator with low sd cannot screen species well. Tissue with a low cold hardiness is more sensitive to freezing damage than a tissue with greater cold hardiness. From the present results, when choosing a single stem tissue as an indicator for screening woody species, the xylem should be considered first followed by the phloem. The cambium and pith were not suitable. The cambium is critical to plant survival (Pellett and Heleba, 1998), but the TTCLT50 for all species was lower than the extreme minimum temperature (−37 °C), and the sd was lowest among the different tissues (Table 4), thus the cambium was not suitable as an indicator of cold hardiness for screening species. TTC staining of the pith was weak and thus the pith was also unsuitable for screening species. The TTC staining and special LT50 was also affected by freezing and TTC test procedure such as time length of freezing test temperature. Some authors provided a similar recommendation to use the xylem as an indicator of cold hardiness using the TTC method (Nesbitt et al., 2002; Pellett and Heleba, 1998); however, the recommendation was based on discoloration of 1-year-old stems of deciduous plants, because the phloem and cambium were difficult to discern with the naked eye or at low magnification and the xylem was easier to observe. Some authors also found the xylem possesses less cold hardiness than the phloem in winter (Einhorn et al., 2011; Parker, 1962).
Estimation of cold hardiness by ELLT50 and TTCLT50.
The ELLT50 showed a higher sd than any tissue TTCLT50 (Table 4). That meant the cold hardiness of test species estimated by ELLT50 showed higher variation than that estimated by TTCLT50. ELLT50 was suitable for screening species as cold hardiness indicator. In field practice experience, ELLT50 fit the growing state of conifer species well. The cold hardiness estimated by ELLT50 showed P. sylvestris and P. asperata could tolerate much lower temperature than J. rigida (Table 1), and the former two species grow much better than the latter species that live in similar environmental conditions in the Bashang region.
ELLT50 represents the lethal temperature at which 50% of electrolyte leakage occurs (Fiorino and Mancuso, 2000), and TTCLT50 means the lethal temperature at which 50% of tissues are damaged (Fiorino and Mancuso, 2000) by visual assessment of staining in the targeted tissue. ELLT50 is associated with erratic function or total dysfunction of cell membranes after freezing, whereas TTCLT50 is associated with the ability of tissue to respire after exposure to cold stress (Soloklui et al., 2012). Steffen et al. (1989) found the function of cell membranes is altered earlier than is the respiratory function of tissue under freezing stress in studying two potato species. Initial alteration of cell membranes is reversible (Palta et al., 1982). This study showed the freezing tolerance of cell membranes was generally lower than that of the respiratory systems in the cambium and phloem. The freezing tolerance measured by EL and by function of respiratory systems in the xylem generally showed no significant difference.
Conclusions
This study mainly showed the cold hardiness differences of woody species evaluated by ELLT50 and TTCLT50. Based on results obtained in this study, when choosing cold hardiness of single stem tissue as an indicator for screening woody species, we recommended considering the xylem first, followed by the phloem. The cambium and pith were not suitable. Using M percentage in the CMYK color system by Adobe Photoshop software could improve the accuracy of TTC staining estimation. The cold hardiness estimated by ELLT50 was more suitable as an indicator for screening woody species than that of stem tissue in winter estimated by the TTCLT50.
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