
==== Front
PLoS One
PLoS One
plos
PLOS ONE
1932-6203
Public Library of Science San Francisco, CA USA

10.1371/journal.pone.0306914
PONE-D-23-42629
Research Article
Biology and Life Sciences
Plant Science
Plant Anatomy
Vascular Bundles
Xylem
Biology and Life Sciences
Physiology
Plant Physiology
Vascular Bundles
Xylem
Biology and Life Sciences
Plant Science
Plant Physiology
Vascular Bundles
Xylem
Biology and Life Sciences
Cryobiology
Cold Hardiness
Biology and Life Sciences
Organisms
Eukaryota
Plants
Fruits
Peaches
Biology and Life Sciences
Agriculture
Animal Products
Honey
Biology and Life Sciences
Nutrition
Diet
Food
Honey
Medicine and Health Sciences
Nutrition
Diet
Food
Honey
Biology and Life Sciences
Organisms
Eukaryota
Plants
Trees
Peach Trees
Research and Analysis Methods
Chemical Characterization
Temperature Analysis
Biology and Life Sciences
Cell Biology
Cellular Structures and Organelles
Cell Membranes
Biology and Life Sciences
Plant Science
Plant Anatomy
Buds
Analyzing cold hardiness (Based on DTA) of one-year-old branches of peaches
Analyzing cold hardiness (Based on DTA) of one-year-old branches of peaches
https://orcid.org/0000-0001-6769-1692
Li Yonghong Conceptualization Data curation Methodology Project administration Visualization Writing – original draft Writing – review & editing 1
Li Jie Data curation Formal analysis Investigation Methodology Writing – original draft 1
Wang Zhaoyuan Data curation Investigation Methodology 1
Liu Guojian Investigation Methodology Resources Writing – original draft Writing – review & editing 1
Wang Yu Data curation Investigation 1
Chang Ruifeng Data curation 1
Chen Hu Data curation 1
Tian Qihang Data curation Investigation Resources Writing – original draft Writing – review & editing 1 *
Wang Xiaodi Data curation Investigation Methodology Project administration Writing – original draft Writing – review & editing 2 *
1 Research Institute of Pomology of CAAS, Xingcheng, Liaoning, China
2 Changli Fruit Institute, Hebei Academy of Agriculture and Forestry Sciences, Changli, Hebei, China
Fu Guanfu Editor
China National Rice Research Institute, CHINA
Competing Interests: The authors received funding from Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Germplasm Resources Utilization), Ministry of Agriculture,P.R.China (No. NYZS202305) and the Fundamental Research Funds for Hebei Academy of Agriculture and Forestry Sciences(No. 2023020102). This does not alter their adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development, or marketed products associated with this research to declare.

* E-mail: liyonghongpeng@163.com (QT); wangxiaodi@caas.cn (XW)
17 9 2024
2024
19 9 e030691418 12 2023
24 6 2024
© 2024 Li et al
2024
Li et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we conducted a low-temperature exothermic (LTE) investigation on 1-year-old (1a) branches of sixteen peach cultivars through a differential thermal analysis (DTA) procedure. We used a three-point approach to determine the lethal injury temperature (LT-I) of the xylem, the LTE correlation indexes, and the subordinate function value method were applied to compare cold hardiness of sixteen peach varieties. The results showed that the slope of the LT-I for the xylem of sixteen peach cultivars was different, and the LTE indexes were significantly different. Among all the studied varieties, the cold hardiness was strongest in Donghe No.1, followed by Wangjiazhuangmaotao No.2 and Hunchun. Qiuyan and Yanhong are second, and belong to the cold-resistant type; Qiuyi, Okubo, Zhongnongjinhui, and Chunmei, exhibited medium cold hardiness. Zhongtaohongyu, Spring snow, Yufei, and Zhongyou No.8 varieties exhibited low hardiness; while the 21st century, Golden Honey No. 1 and Zhonghuashoutao have the worst cold hardiness and are the weakest cold-hardiness types. In addition, the injury degrees of xylem from LT-I analysis were significantly related to the browning rates (BR) and electrolytic leakage (EI) from traditional low temperature freezing analysis. It is demonstrated that the LTE analysis is a simple, accurate, and practical method for identifying the cold hardiness of 1a branches of peach.

http://dx.doi.org/10.13039/501100014962 Key Laboratory of Biology and Genetic Improvement of Horticultural Crops NYZS202305 https://orcid.org/0000-0001-6769-1692
Li Yonghong Key R&D Projects of Hebei Province 21326310D Liu Guojian Technology Innovation Special Project of Hebei Academy 2022KJCXZX-CGS-6 https://orcid.org/0000-0001-6769-1692
Li Yonghong Modern Agricultural Industrial Technology System of Hebei Province HBCT2023130205 Wang Zhaoyuan China Agriculture Research System of MOF and MARA CARS-30-Z-02 Chen Hu Key R&D Projects of Hebei Province 2023020102 Tian Qihang This work was supported by Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Germplasm Resources Utilization), Ministry of Agriculture,P.R.China (No. NYZS202305) and the Fundamental Research Funds for Hebei Academy of Agriculture and Forestry Sciences(No. 2023020102), Technology Innovation Special Project of Hebei Academy (No. 2022KJCXZX-CGS-6), Key R&D Projects of Hebei Province (No.21326310D), Modern Agricultural Industrial Technology System of Hebei Province (No. HBCT2023130205) and China Agriculture Research System of MOF and MARA (No. CARS-30-Z-02). The funders played the important role in study design, data collection and analysis, decision to publish, and preparation of the manuscript. Data AvailabilityAll relevant data are within the manuscript.
Data Availability

All relevant data are within the manuscript.
==== Body
pmcIntroduction

Peach (Prunus persica (L.) Batsch), a nutrient-rich fruit, has been extensively distributed worldwide [1, 2]. However, its growth, development, and distribution are restricted by low temperature, a crucial constraint [3]. There are many reports (national and international) on the identification methods and cold hardiness physiology of peach. The methods of EI are used to compare the cold hardiness between varieties by combining the contents of physiological and biochemical indexes such as free water, MDA, soluble sugars, soluble proteins and proline, germination rate, BR, and recovery growth methods [4–7]. It is time-consuming, laborious, and has many influencing factors.

Since the 1970s, the low temperature exothermic (LTE) technique has been improved considerably, thus allowing the comparison of cold hardiness across various plant varieties. For LTE examination, recording, and analysis, in the LTE technique, ice crystals are formed in tissues at low temperatures with a PC-assisted procedure of differential thermal analysis (DTA), as well as further cold hardiness evaluation of plant tissues [8, 9]. In previous studies, data analysis was performed by the half-lethal temperature (LT50) or LT10 method [10–13], which was unable to sufficiently reflect the cold hardiness trait of the different varieties. Later, Gu et al. [14] performed a cold hardiness assessment of buds by using slope Qlt and LT50, which were more comprehensive and accurate, and provided a new way for analyzing the cold hardiness of branches. The comprehensive evaluation of plant cold hardiness has been performed effectively by the subordinate function method for poplar [15], grape [16], and apple [17].

Flower buds and trunk tissues are susceptible to freezing or frost damage [18]. Minas et al. [19] have revealed that DTA accurately predicts mid- and late-winter cold hardiness and damage of peach floral buds, however, it loses the capacity to determine ice nucleation events as bud development advances in spring. Currently, there is no report on the use of the LTE method to analyze the cold-hardiness of the 1a branches of peach varieties. In this study, using dormant branches of 16 peach varieties, the exothermic characteristics and LTE-related indexes of each variety were analyzed by the LTE method, and the regression analysis was performed for the xylem lethal injury temperature (LT-I). The cold-hardiness of 1a branches of 16 peach varieties was comprehensively evaluated by the membership function method combined with LTE-related indexes, and the results were verified by the conductivity method and the tissue browning method. To provide technical support for cold-hardiness identification of peach germplasm, cold hardiness breeding, and planting regionalization of existing varieties, an accurate and reliable method for cold hardiness identification of peach was established.

Materials and methods

Experimental materials

Trials were performed within the peach orchard in Changli, China (39°45′N, 119°12′E). It is an area with a continental monsoon type of climate, with four seasons. The Prunus persica cultivars used in this study included Wangjiazhuangmaotao No.2, Hunchun, Donghe No.1, Qiuyan, Yanhong, Qiuyi, Okubo, Zhongnongjinhui, Chunmei, Zhongtaohongyu, Spring snow, Yufei, Zhongyou No.8, 21st century, Golden honey No.1, and Zhonghuashoutao.

Experimental instruments

The DTA system used in this study comprised a data acquiring unit (fabricated by the Mechanical and Electronic Engineering School, Shandong Agricultural University, China) and programmable temperature incubators (GDTS-2252, Wuxi Jingchuang Technology Co., Ltd, China). The incubators could be operated between –70 and +150°C and were 600 mm × 750 mm × 500 mm in size. The data acquiring unit included 27 Peltier thermoelectric modules (40 mm × 40 mm × 15 mm). This unit was responsible for sensing freezing exotherms and converting them into voltage outputs. Additionally, it could also record temperature variation during exothermic procedures through a pt100 sensor, with an accuracy reaching 0.1%.

Experimental methods

LTE determination method

The 1a branches of the different varieties were cut with a pair of scissors into 2.0-cm-long sections that were 0.8–1.0 cm in diameter. The sections were each placed into individual thermoelectric modules (triplicates) and pressed using a plastic foam board (600 mm × 750 mm × 500 mm) to maintain adequate contact with the thermoelectric modules. Later, the temperature was reduced as follows: 1 h at room temperature to 4°C, 1 h at 0°C, 11 h from 0°C to –44°C, and 1 h at –44°C [1, 13, 20, 21].

Determination of EI and BR

’Wangjiazhuangmaotao No.2’, ’zhongnongjinhui’ and ’Chunmei’ branches were collected to detect the electrolytic leakage (EI) and the browning rate (BR). On 5th January 2020, branches of equal diameter (0.8–1.0 cm) and length (25–30 cm) (from the same batch of samples as LTE) were harvested from all sides of the 7-year-old trees. The branches were cleaned with distilled water and separated into six groups, each of which was wrapped with an adequate amount of gauze and placed in a plastic bag. The temperature treatments provided were 4°C (control), –23°C, –27°C, –31°C, –35°C and –39°C, with 12 h of treatment time.

For EI estimation, the method used was as described in a previous study [22].

BR was determined by the tissue browning method [18]. Twenty branches of each variety were examined. From actual observations and the browning area of the secondary xylem, a five-grade classification system was adopted to classify the freezing injury. The categories were as follows: the browning area of Grade 0 xylem was 0–3%, Grade 1 was 4–30%, Grade 2 was 31–50%, Grade 3 was 51–75%, and Grade 4 was 76–100%.

Data analysis

The SPSS 20.0 software (SPSS Inc., Chicago, IL, U.S.A.) was used for all statistical analyses; analysis of variance and correlation analyses were performed. All data are expressed as the mean ±standard error; the significance level was set at P < 0.01. The root cold-hardiness analysis was used to perform the LT-I regression [23]. The LSD approach was adopted to perform analysis of variance (ANOVA) for the lethal temperature coefficient (LT50) and the slope (Qlt), shown in Table 1. The severity of the injury of all cultivars was estimated based on the LT-I at various temperatures (Table 2). The subordinate function value analysis was performed for evaluating the cold hardiness of the 1a branches comprehensively using the formula: R(Xi)=1‐(Xi‐Xmin)/(Xmax‐Xmin).

10.1371/journal.pone.0306914.t001 Table 1 LT-I equation of 1-year-old branch xylem of 16 peach varieties.

Variety	LT-I	R2	Qlt	LT50/°C	LT0-LT100/°C	The difference value between LT0 and LT100/°C	
    Zhongtaohongyu	y = -7.65x - 187.05	0.967	-7.65AB	-30.99B	-24.63–37.31	12.68	
    Qiuyan	y = -10.57x - 294.43	0.991	-10.57C	-32.59DEF	-27.90–37.28	9.38	
    Hunchun	y = -11.27x - 324.23	0.995	-11.27C	-33.21EF	-28.79–37.62	8.83	
21st Century	y = -6.55x - 137.58	0.996	-6.55A	-28.64A	-21.05–36.27	15.22	
Spring Snow	y = -7.41x - 179.87	0.995	-7.41AB	-31.02B	-24.32–37.76	13.44	
    Zhonghuashoutao	y = -6.47x - 146.30	0.964	-6.47A	-30.34BC	-22.40–37.29	14.89	
    Qiuyi	y = -7.85x - 196.83	0.990	-7.85AB	-31.44BCD	-25.50–38.11	13.20	
Golden honey No.1	y = -6.57x - 144.70	0.988	-6.57AB	-29.63A	-22.02–37.25	15.23	
Okubo	y = -6.46x - 159.43	0.983	-6.46A	-32.42BCD	-25.53–38.34	12.81	
    Yanhong	y = -7.27x - 190.78	0.931	-7.27AB	-33.12BCD	-27.53–40.34	12.81	
    Yufei	y = -7.99x -197.66	0.989	-7.99B	-31.00B	-24.81–37.19	12.38	
Wangjiazhuangmaotao No.2	y = -12.31x - 370.48	0.974	-12.31D	-34.16EF	-30.54–38.46	7.92	
     Zhongyoutao No.8	y = -7.59x - 185.44	0.943	-7.59B	-31.02B	-24.81–37.22	12.41	
    Donghe No.1	y = -10.54x - 304.17	0.992	-10.54C	-33.60F	-28.90–38.31	9.41	
    Zhongnongjinhui	y = -8.20x - 215.98	0.988	-8.20B	-32.44CDE	-26.41–38.46	12.05	
    Chunmei	y = -8.03x - 207.56	0.975	-8.03AB	-32.07CDE	-28.17–37.98	9.81	
Note: LT-I indicated the regression line of the lethal injury temperature; R2:The coefficients of determination; Qlt indicated the slope of LT-I; LT50 indicated the half lethal temperature; LT0-LT100/°C indicated temperature range corresponding to 0–100% injury degree severity; The data within a column followed by different big letters indicate significant difference (p<0.01).

10.1371/journal.pone.0306914.t002 Table 2 Computation of 16 peach varieties at different low temperatures.

Variety	-17°C	-19°C	-21°C	-23°C	-25°C	-27°C	-29°C	-31°C	-33°C	-35°C	-37°C	-39°C	-41°C	
Zhongtaohongyu	0	0	0	0	4.20	19.50	34.80	50.10	65.40	80.70	96.00	100	100	
     Qiuyan	0	0	0	0	0	0	12.10	33.24	54.38	75.52	96.66	100	100	
    Hunchun	0	0	0	0	0	0	2.60	25.14	47.68	70.22	92.76	100	100	
21st Century	0	0	0	13.07	26.17	39.27	52.37	65.47	78.57	91.67	100	100	100	
    Spring Snow	0	0	0	0	5.38	20.2	35.02	49.84	64.66	79.48	94.3	100	100	
    Zhonghuashoutao	0	0	0	2.51	15.45	28.39	41.33	54.27	67.21	80.15	93.09	100	100	
    Qiuyi	0	0	0	0	0	15.12	30.82	46.52	62.22	77.92	93.62	100	100	
    Golden honey No.1	0	0	0	6.41	19.55	32.69	45.83	58.97	72.11	85.25	98.39	100	100	
    Okubo	0	0	0	0	2.07	14.99	27.91	40.83	53.75	66.67	79.59	92.51	100	
    Yanhong	0	0	0	0	0	5.51	20.05	34.59	49.13	63.67	78.21	92.75	100	
    Yufei	0	0	0	0	2.09	18.07	34.05	50.03	66.01	81.99	97.97	100	100	
Wangjiazhuangmaotao No.2	0	0	0	0	0	0	0	11.13	35.75	60.37	84.99	100	100	
    Zhongyoutao No.8	0	0	0	0	4.31	19.49	34.67	49.85	65.03	80.21	95.39	100	100	
    Donghe No.1	0	0	0	0	0	0	1.49	22.57	43.65	64.73	85.81	100	100	
    Zhongnongjinhui	0	0	0	0	0	5.42	21.82	38.22	54.62	71.02	87.42	100	100	
Chunmei	0	0	0	0	0	9.25	25.31	41.37	57.43	73.49	89.55	100	100	

Here, R is the value of the subordinate function; Xi is the measured value of a certain index; Xmin and Xmax represent the minimum and maximum values, respectively, of the index in all resources tested. The value of the subordinate function of each LTE index was calculated by the above-mentioned formula, and the mean value of the LTE index of each variety was taken as the mean value of the subordinate degree. Based on the average subordinate degree, the cold hardiness was divided into five grades: 0.70–1.00 was high hardiness (HH), Grade 1; 0.60–0.69 was hardiness (H), Grade 2; 0.40–0.59 was medium hardiness (MH), Grade 3; 0.30–0.39 was low hardiness (LH), Grade 4; 0–0.29 was the weakest grade (WH), Grade 5.

Results

Construction of the LT-I regression equation and estimation of Qlt & LT50

The DTA system was used to obtain the LTE pattern of the peach branches. The continuous cooling process revealed two exothermic peaks, according to the exothermic characteristics. The first exothermic peak observed above –10°C was attributed to a high-temperature exothermic process (Fig 1). The second exothermic peak occurred between –25°C and –40°C (Fig 1), indicating the presence of LTE characteristics. The first exothermic process did not cause any freezing injury to the branches, indicating that the exothermic treatment did not cause freezing injury associated with the freezing of intercellular water and the xylem conduit gravity water. The second exothermic process was at a lower temperature, which was directly related to freezing injury. This might have been due to the death of xylem parenchyma cells caused by deep super cooled water freezing. It was observed that the secondary exothermic process occurred in the separated xylem, and its exotherm resembled that of the branches. Freezing injury of the xylem was directly caused by the freeze of super cooled water. However, no second exotherm in the bark, suggesting that freezing damage is minimally influenced by the peach skin.

10.1371/journal.pone.0306914.g001 Fig 1 Typical differential thermal analysis (DTA) profiles of low temperature exotherms (LTE) and high temperature exotherms (HTE) of peach 1a branch.

Point A shows the initial freezing point of xylem. Point B shows the complete xylem exothermic point. Point C shows that xylem exothermic reached the peak. In Fig 1, Point A represents the initial freezing point of xylem, denoted as LT0, indicating 0% xylem injury severity. Besides, as shown in Table 1, Point B indicates the complete exothermic point of xylem, denoted as LT100, representing 100% xylem injury severity. There were significant differences in the low-temperature exothermic temperature of the different varieties. According to the data in Table 1, the ranking of the plant varieties for the LT50 of xylem was as follows: 21st Century > Golden honey No.1 > Zhonghuashoutao > Zhongtaohongyu > Yufei > Zhongyoutao No.8 = Spring snow > Qiuyi > Chunmei > Okubo > Zhongnongjinhui > Qiuyan > Yanhong > Hunchun > Donghe No.1 > Wangjiazhuangmaotao No.2. Also, it is indicated that the LT50 of the 21st Century and Golden honey No.1 varieties was higher than -30°C, and that of the Wangjiazhuangmaotao No.2 variety was below-34°C; the rest of the varieties had LT50 values between-30°C and-33°C. The difference of LT50 between the 21st Century and Wangjiazhuangmaotao No.2 varieties was 5.52°C (Table 1).

To plot the xylem graphs (Table 1), a three-point regression analysis was investigated. The points on the graph, namely LT0, LT100, and LT50 corresponded to the initial freezing point A, final freezing exothermic B, and peak C of the xylem as depicted in Fig 1. Regression coefficient (Qlt), which is the slope of the LT-I graph, indicates the intensification of the severity of branch injury for the drop in temperature by 1°C. With an increase in the absolute Qlt value, the varieties become more vulnerable to the drop in temperature and freezing injury. According to the data in Table 1, the ranking of the xylem Qlt of all varieties were as follows: Wangjiazhuangmaotao No.2 < Hunchun < Qiuyan < Donghe No.1 < Zhongnongjinhui < Chunmei < Yufei < Zhongtaohongyu < Zhongyoutao No.8 < Qiuyi < Spring snow < Yanhong < Golden honey No.1 < 21st Century < Zhonghuashoutao < Okubo. The correlation between Qlt and LT0 –LT100 was significantly positive (correlation coefficient of 0.918). The 1a branch temperature range of the different peach varieties was found to be broad, and the ability to resist the damage of cooling was high (Table 2).

Comparison of cold hardiness of different peach varieties

Gao Zhen (Gao et al., 2014) conducted an estimation and verification, revealing two distinct types of LT-I in the case of root cold hardiness: intercepted and non-intercepted types, each further classified into three subtypes. A similar classification was observed for the cold hardiness of branches (Fig 2).

10.1371/journal.pone.0306914.g002 Fig 2 The crossing of the regression line of the lethal injury temperature (LT-I) of different peach varieties 16 cultivars were divided to four groups: A, B, C, D.

In terms of the cold hardiness of non-intercepted varieties, three sub types were identified. For the first subtype, there is no significant difference between Qlt and LT50, which shows that the cold hardiness of the branches of the two peach varieties is basically the same. Such as: Zhongyoutao No.8 and Yufei (Table 1 and Fig 2B and 2D), Qiuyi and Yanhong (Table 1 and Fig 2C). The second type (Qlt) showed no significant difference, but LT50 exhibited some variations. For instance, Hunchun and Donghe No.1 (Table 1 and Fig 2B), Zhongtaohongyu and Qiuyi (Table 1 and Fig 2C), Zhongnongjinhui and Zhongyoutao No.8 (Table 1 and Fig 2D). The performance of the two peach varieties in LT-I were parallel. The lower the LT50, the stronger the cold hardiness was seen in the branches. The third type, Qlt and LT50 showed significant differences, the lower the LT50, the stronger the cold hardiness of the 1a branches was seen. Additionally, a smaller Qlt indicated less injury when the temperature drops by the same degree r (Table 2), such as that in 21st Century and Okubo (Table 1 and Fig 2A), Hunchun and Donghe 1 (Table 1 and Fig 2B), and Qiuyan and Donghe 1 (Table 1 and Fig 2D), all conform to this type.

Comparison of the cold hardiness of LT-I intercepted varieties was conducted. The presence of a significant difference between Qlt and LT50 was used to compare cold hardiness, among the intercepted varieties, Similar Qlt and LT50 values indicated no difference in cold hardiness between the two varieties. On the contrary, a significant difference between Qlt and LT50 indicated that cold hardiness should be assessed before and after the interception point. Gao (2014) classified the intercepted LT-I values of roots into three subtypes depending on the LT50 occurrence relative to the interception point. For branch comparisons, there could be three subtypes as well. For the first subtype, the intersection point is before LT50. The interception point (–30.67°C) of the LT-I of the Spring snow and Yufei varieties was noted before the LT50 (Table 1 and Fig 2B). At temperatures exceeding –30.67°C, Yufei exhibited stronger cold-hardiness than Spring snow, while Spring snow showed better cold-hardiness below that temperature. Additionally, Qiuyan and Chunmei also belonged to this subtype (Table 1 and Fig 2D). In the second subtype, the intersection point (-34.20°C) of Qiuyan and Zhongnongjinhui occurred after LT50 (Table 1 and Fig 2D). Zhongnongjinhui exhibited stronger cold-hardiness above -34.20°C, while Qiuyan showed better cold-hardiness below that temperature. For the third subtype (not displayed), the intersection point and LT50 were coincident, which did not occur in this study.

Comparison of injury degree of different peach varieties under programmed cooling

By the LT-I regression equation, the degree of injury under different temperatures was determined (Table 1). Herein., the degree of injury to branches less than 0% was recorded as 0, and the degree of injury greater than 100% was recorded as 100 (Table 2). The degree of injury to the branches of the 21st Century, Golden honey No.1 and Zhonghuashoutao varieties were 13.07%, 6.41%, and 2.51%, respectively, at –23°C, while that of Wangjiazhuangmaotao No.2 was 11.13% at –31°C. The degree of injury of Qiuyan, Hunchun, and Donghe No.1 was 33.24%, 25.14%, and 22.57%, respectively, at –31°C. It is generally believed that branch injuries exceeding 20% have an impact on production. These findings revealed that the low cold-hardiness varieties of 21st century, Golden honey No.1, and Zhonghuashoutao injuries exceeding 20% between –25°C and –27°C, while high cold-hardiness varieties experienced injuries exceeding 20% between –31°C and –33°C. Furthermore, injuries exceeding 20% was observed in the varieties between high hardiness and low hardiness between –27°C and –29°C. The temperature-sensitivity range of the different cold-hardiness peach varieties was different, for example, the low cold-hardiness varieties (21st Century, Golden honey No.1, Zhonghuashoutao) showed sensitivity between –25°C and –27°C, the high cold-hardiness varieties showed sensitivity between –31°C and –33°C, and the medium cold-hardiness varieties showed sensitivity between –27°C and –29°C (Table 2).

When the winter temperature dropped below-33°C, most of the varieties had frostbites or even died and the degree of injury reached 50%-70%. However, more than half of the 1a branches of high cold-hardiness varieties could safely overwinter, especially Wangjiazhuangmaotao No.2, Donghe No.1, and Hunchun, whose degrees of injury were 35.75%, 43.65%, and 47.68%, respectively (Table 2).

Comprehensive assessment of LTE data using the subordinate function method

As shown in Table 3, the subordinate function method was used for analyzing the LTE data. Varieties with higher function values demonstrated stronger cold hardiness. Among the branches, the 21st Century, Golden honey No.1, and Zhonghuashoutao exhibited the weakest cold-hardiness (WH). While Zhongtaohongyu, Spring snow, Yufei, and Zhongyoutao No.8 had low cold-hardiness (LH). Qiuyi, Okubo, Zhongnongjinhui, and Chunmei had medium cold-hardiness (MH), Qiuyan and Yanhong had good cold-hardiness (H), and Wangjiazhuangmaotao No.2, Donghe No.1, and Hunchun had the highest cold-hardiness (HH).

10.1371/journal.pone.0306914.t003 Table 3 Identification results of cold resistances of 16 peach varieties.

Variety	Subordination value	Cold-hardiness type	
Qlt	Z0/°C	LT50/°C	Z100/°C	Average	
Zhongtaohongyu	0.20	0.38	0.43	0.26	0.32	LH	
    Qiuyan	0.70	0.72	0.71	0.25	0.60	H	
    Hunchun	0.82	0.82	0.83	0.33	0.70	HH	
21st Century	0.02	0.00	0.00	0.00	0.00	WH	
Spring Snow	0.16	0.34	0.43	0.37	0.33	LH	
Zhonghuashoutao	0.00	0.14	0.31	0.25	0.18	WH	
Qiuyi	0.24	0.47	0.51	0.45	0.41	MH	
    Golden honey No.1	0.02	0.15	0.18	0.32	0.17	WH	
    Okubo	0.00	0.47	0.68	0.51	0.42	MH	
    Yanhong	0.14	0.68	0.81	1.00	0.66	H	
Yufei	0.26	0.40	0.43	0.23	0.33	LH	
Wangjiazhuangmaotao No.2	1.00	1.00	1.00	0.54	0.88	HH	
    Zhongyoutao No.8	0.19	0.40	0.43	0.23	0.31	LH	
    Donghe No.1	0.70	0.83	0.90	0.50	0.73	HH	
Zhongnongjinhui	0.30	0.56	0.69	0.54	0.52	MH	
Chunmei	0.27	0.75	0.62	0.42	0.52	MH	
Note: Qlt: the slope of xylem LT-I injury of 1-year-old branches; Z0: the freezing point of xylem, that is, the injury degree of xylem is 0; Z100: the xylem freezes completely, that is, the injury degree of xylem is 100%; LT50: the half lethal temperature of branches calculated according to the regression equation LT-I in Table 1.

The relationship between the LTE subordination value of a branch and its BR and EI

Based on the subordinate function values (Table 3), three representative varieties with cold hardiness ranks of strong, moderate, and weak were selected from the studied varieties (Table 4 and Fig 3). The EI and BR of these varieties were determined, the degree of damage to the xylem was calculated, and correlation analysis was performed.

10.1371/journal.pone.0306914.g003 Fig 3 Freezing injury of xylem of 1a branches of peach after low temperature treatment.

A: Wangjiazhuangmaotao No.2 B: Zhongnongjinhui C: Chunmei.

10.1371/journal.pone.0306914.t004 Table 4 Comparison of LTE and traditional indexes of three peach varieties.

Temperature /°C	(LTE) Injury degree of xylem/%	(Traditional indexes) Browning rate of xylem /%	(Traditional indexes) Injury degree determined by
electrical conductivity/%	
Wangjiazhuangmaotao No.2	Zhongnongjinhui	Chunmei	Wangjiazhuangmaotao No.2	Zhongnongjinhui	Chunmei	Wangjiazhuangmaotao No.2	Zhongnongjinhui	Chunmei	
-23°C	0.00	0.00	0.00	0.00	0.00	0.00	28.70	29.05	28.93	
-27°C	0.00	5.42	9.25	0.00	20.00	26.00	30.56	31.64	32.26	
-31°C	11.13	38.22	41.37	4.00	50.00	88.00	36.41	48.27	50.41	
-35°C	60.37	71.02	73.49	72.00	96.00	98.00	59.87	71.24	72.35	
-39°C	100.00	100.00	100.00	100.00	100.00	100.00	74.22	74.35	78.00	

Different peach cultivars exhibited varying degrees of browning in the xylem of the 1a branch under low-temperature treatment. Wangjiazhuangmaotao No.2, Zhongnongjinhui, and Chunmei branches showed no damage at -23°C (Table 4 and Fig 3), classified as Grade 0 according to the BR classification for freeze injury. At –27°C, the high hardiness variety Wangjiazhuangmaotao No.2 had no change, but the medium cold-hardiness varieties (Zhongnongjinhui and Chunmei) had a xylem browning rate of 20%, and the freezing injury was Grade 1, i.e., it was slightly affected by freezing. When the freeze injury level of Wangjiazhuangmaotao No.2 reached Grade 1, the injury level of Zhongnongjinhui and Chunmei reached Grade 3 at –31°C. When the freeze injury level of Wangjiazhuangmaotao No.2 reached Grade 3, the injury level of Zhongnongjinhui and Chunmei reached Grade 4, and the freezing injury was 100%. The results were consistent with the xylem injury data measure in LTE during the cooling process. Correlation analysis of the three peach cultivars showed a positive correlation between BR and injury of xylem, with correlation coefficients were 0.991, 0.999, and 0.907, respectively (Table 5). This indicated that the LTE analysis accurately reflected the cold-hardiness of 1a peach branches.

10.1371/journal.pone.0306914.t005 Table 5 Correlation analysis of LTE and traditional indexes of three peach varieties.

Variety	Index	LTE indexes	Traditional indexes	

Injury degree of xylem	Browning rate of xylem	Injury degree determined by
electrical conductivity	
WangjiazhuangmaotaoNo.2	Injury degree of xylem	1	 	 	
Browning rate of xylem	0.991**	1		
Injury degree determined by
electrical conductivity	0.997**	0.993**	1	
Zhongnongjinhui	Injury degree of xylem	1			
Browning rate of xylem	.999**	1		
Injury degree determined by
electrical conductivity	.950*	.943*	1	
Chunmei	Injury degree of xylem	1			
Browning rate of xylem	.907*	1		
Injury degree determined by
electrical conductivity	.991**	.920*	1	
Note: * Significant at 0.05 levels

** Significant at 0.01 levels.

We analyzed the degree of injury to 1a branches of the three peach cultivars by electrolytic leakage (EI), and the results are shown in Table 4. The association of EI with the severity of xylem injury, calculated according to the LT-I and adequate values of the subordinate function (Table 4), was determined. The results showed that the degree of injury to the xylem was positively correlated with the EI, and the correlation coefficients of the three peach cultivars were 0.997, 0.950, and 0.991, respectively (Table 5), demonstrating that the LTE analysis accurately reflected the cold-hardiness of the 1a peach branches.

Discussion

Analysis of cold-hardiness of the different peach branches examined

The cold hardiness analysis was performed by the LT-I procedure for the 16 studied cultivars. Among the cultivars examined, Wangjiazhuangmaotao No.2, Donghe No.1, and Hunchun had the strongest cold-hardiness (HH), and Qiuyan and Yanhong had strong cold-hardiness (H), while 21st century, Golden honey No.1, and Zhonghuashoutao exhibited the weakest cold hardiness (WH). The inter-variety differences in cold hardiness were primarily due to the variations in the plant genetic traits. Different peach varieties, on exposure to different environments for a prolonged period, developed different genotypes, which led to variations in those traits that helped to adapt to the environment, such as cold hardiness [23]. Additionally, geologic origin and acclimatization are also factors that influence cold hardiness. For example, the cold hardiness of the northern varieties, originating from cold areas, Wangjiazhuangmaotao No.2, Donghe No.1, and Hunchun, was higher than that of the southern varieties, Golden honey No.1 and Zhongyou No.8.

Relationship between BR and cold-hardiness branches

Tissue browning is one of the traditional methods to identify the cold hardiness of plants. Since Levitt (1956) first applied the tissue browning method to study cold-hardiness, significant progress has been made in the improvement of the method, theoretical discussion, and practical application, and it has become a fast and reliable method to study stress physiology [24]. In this study, the results of the correlation analysis of the three peach cultivars showed that the degree of injury to the xylem was positively correlated with the BR of the xylem (Table 4 and Fig 3). Similarly, Mills [21] also proved that bud and cane LTE recorded by the DTA system had a strong correlation, and the results of the severity of injury to cane xylem and phloem from tissue browning tests were similar to those based on the LTE analysis. This indicated that the LTE analysis could accurately reflect the cold-hardiness of peach branches.javascript:void(0);

Relationship between EI and cold-hardiness of branches

One of the primary parts affected by freezing injury in plants is the cell membrane. The injury of low temperature to the membrane can lead to an increase in the EI [3]. Correlation analysis showed that the heat release of the xylem at low temperatures was associated with the permeability of the protoplasm membrane. In this study, the EI of Wangjiazhuangmaotao No.2, Zhongnongjinhui, and Chunmei were measured at different temperatures. It was found that the relative conductivity of Wangjiazhuangmaotao No.2 was lower than that of Zhongnongjinhui and Chunmei at the same temperature (Table 4), which indicated that the cold hardiness of Wangjiazhuangmaotao No.2 was higher than that of Zhongnongjinhui and Chunmei. The degree of injury to the plasma membrane of Wangjiazhuangmaotao No.2 was lower than that in the plasma membrane of Zhongnongjinhui and Chunmei, which was consistent with the degree of injury to the xylem measured in the LTE stage during programmed cooling. This indicated that the low-temperature exotherms of the xylem were related to the permeability of the plasma membrane.

Detection method of cold-hardiness of peach branches

Cold hardiness analysis based on LTE has been extensively conducted in grape research [13, 21, 23], suggesting that it is a reliable analytical technique. In this three-point study, we performed branch LT-I determination on various peach cultivars using LT0, LT50, and LT100 through LTE investigation of cold hardiness, which was faster, simpler, and more accurate than the previous approaches.The results showed that the LTE curve of peach branches in the dormancy period had a single peak, which separated the xylem from the bark. The supercooled water of the separated xylem was similar to that of the branches. It was found that freezing injury to the xylem was directly caused by supercooled water freezing. There was no second exothermic heat in the bark, which indicated that the freezing injury to the bark was not associated with the low-temperature exothermic heat, and the results were similar to previous findings [18, 20]. In conclusion, the cold-hardiness of the 1a peach branches was mainly determined by the xylem. However, the cold-hardiness of some tree species is determined by the phloem, i.e., when the degree of injury to the phloem reaches 100%, the branches freeze to death, for example, in grape and chestnut [20, 25].

Conclusions

In conclusion, the DTA system was utilized to perform LTE test on 1a branches of peach varieties. The application of LT-I regression linear allows for a complete and comprehensive comparison of branch cold hardiness among different varieties, accurate identifying the temperature at which xylem experience frost damage. The test method is simpler, faster and more reliable compared to determining physiological index, making it suitable for evaluating the cold hardiness of branches in different peach varieties. The comparison of LTE composite indices revealed that that Wangjiazhuangmaohuo No.2, Hunchun and Donghe No.1 belong to the high cold resistance type; Qiuyan and Yanhong are the next highest level of cold hardiness, while 21st Century, Golden Honey 1 and Zhonghuashoutao displayed the lowest cold hardiness and the least cold hardiness type among the tested varieties. In addition, the injury degrees of xylem from LT-I analysis were significantly related to the browning rates and electrolytic conductivity from traditional low temperature freezing analysis. Based on the above research results, it is believed that the LTE analysis method is a simple, accurate, and practical method for evaluating the cold resistance of peach branches, which can be used for identifying the cold hardiness of peach germplasm resources in the future.

We are grateful for the technical support provided by Bin Han and Jicheng Han.

10.1371/journal.pone.0306914.r001
Decision Letter 0
Fu Guanfu Academic Editor
© 2024 Guanfu Fu
2024
Guanfu Fu
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
14 Mar 2024

PONE-D-23-42629Analyzing Cold Hardiness (Based on DTA) of One-Year-Old Branches of PeachesPLOS ONE

Dear Dr. Li,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Apr 28 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Guanfu Fu, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

3. Thank you for stating in your Funding Statement: 

This work was supported by Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Germplasm Resources Utilization), Ministry of Agriculture,P.R.China (No. NYZS202305) and Technology Innovation Special Project of Hebei Academy (No. 2022KJCXZX-CGS-6), Key R&D Projects of Hebei Province (No. 21326310D), Modern Agricultural Industrial Technology System of Hebei Province (No. HBCT2023130205) and China Agriculture Research System of MOF and MARA (No. CARS-30-Z-02).

Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. 

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

This work was supported by Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Germplasm Resources Utilization), Ministry of Agriculture,P.R.China (No. NYZS202305) and Technology Innovation Special Project of Hebei Academy (No. 2022KJCXZX-CGS-6), Key R&D Projects of Hebei Province (No. 21326310D), Modern Agricultural Industrial Technology System of Hebei Province (No. HBCT2023130205) and China Agriculture Research System of MOF and MARA (No. CARS-30-Z-02).

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

This work was supported by Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Germplasm Resources Utilization), Ministry of Agriculture,P.R.China (No. NYZS202305) and Technology Innovation Special Project of Hebei Academy (No. 2022KJCXZX-CGS-6), Key R&D Projects of Hebei Province (No. 21326310D), Modern Agricultural Industrial Technology System of Hebei Province (No. HBCT2023130205) and China Agriculture Research System of MOF and MARA (No. CARS-30-Z-02).

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

6. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

7. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments

The authors selected Wangjiazhuangmaotao No.2, Zhongnongjinhui, and Chunmei as three representative varieties with strong, moderate, and weak cold hardiness ranks to determine EI and BR. However, Table 3 showed that Zhongnongjinhui and Chunmei are both medium cold-hardiness (MH) variety. In the subsequent results, there was a difference in data of BR between Zhongnongjinhui and Chunmei, both of which were MH variety. Does this indicat that the identification of cold hardness through LTE investment of is not as precise? Please explain in detail.

Minor points

1. Please review the manuscript for punctuation errors throughout the text, e.g. line 88 "... electrical leakage (EI)]".

2. Please review the manuscript for the consistency of font size, e.g. line 96 "A total of …", and line 164 "…hardiness…".

3. Please carefully check the reference format and ensure consistency, e.g. line 346 "Am. J. Enol. Vitic", and line 352 "Annals of Botany".

Reviewer #2: In this manuscript, the authors demonstrated the validity of LTE analysis for cold hardiness of peach branches by analyzing the exothermic behavior, brown rates, and the electrolytic leakage of one-year-old branches of sixteen peach cultivars under low temperature conditions. This work could be accepted after minor revisions. Other questions were shown below:

1. There are multiple formatting errors throughout the manuscript, such as the incorrect use of spaces in lines 34, 66, 74, 77, etc.

2. Line 80: "the LTE determination method involved cutting" should not be formatted in bold.

3. Fig 1: Graphing error, there is no point C in the diagram.

4. Line 140: “There” should be “there”.

5. Line 256: An “of” should be removed.

6. Line 260: “wiht” should be “with”.

7. Line 278: “siince” should be “since”.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment Submitted filename: Review comments.docx

10.1371/journal.pone.0306914.r002
Author response to Decision Letter 0
Submission Version1
8 May 2024

Response to the comments of the academic editor and the reviewers

Manuscript Number: PONE-D-23-42629

Manuscript Title: Analyzing Cold Hardiness (Based on DTA) of One-Year-Old Branches of Peaches

Dear Editors:

Thanks for your kind comments. We have carefully read the whole manuscript and revised it in accordance with the academic editor and the reviewers’ comments. The corrections and additions are highlighted in red color in manuscript. We list the reply as follows:

Guanfu Fu, Ph.D

Academic Editor

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Reply:We have revised the manuscript according to PLOS ONE's style requirements.

2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

Reply:

No permits statement

We are a non-profit scientific research institutions and do not need to provide a permit.

3. Thank you for stating in your Funding Statement:

This work was supported by Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Germplasm Resources Utilization), Ministry of Agriculture,P.R.China (No. NYZS202305) and Technology Innovation Special Project of Hebei Academy (No. 2022KJCXZX-CGS-6), Key R&D Projects of Hebei Province (No. 21326310D), Modern Agricultural Industrial Technology System of Hebei Province (No. HBCT2023130205) and China Agriculture Research System of MOF and MARA (No. CARS-30-Z-02).

Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

Reply:We have submitted our amended Funding Statement in cover letter.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

This work was supported by Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Germplasm Resources Utilization), Ministry of Agriculture,P.R.China (No. NYZS202305) and Technology Innovation Special Project of Hebei Academy (No. 2022KJCXZX-CGS-6), Key R&D Projects of Hebei Province (No. 21326310D), Modern Agricultural Industrial Technology System of Hebei Province (No. HBCT2023130205) and China Agriculture Research System of MOF and MARA (No. CARS-30-Z-02).

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

This work was supported by Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Germplasm Resources Utilization), Ministry of Agriculture,P.R.China (No. NYZS202305) and Technology Innovation Special Project of Hebei Academy (No. 2022KJCXZX-CGS-6), Key R&D Projects of Hebei Province (No. 21326310D), Modern Agricultural Industrial Technology System of Hebei Province (No. HBCT2023130205) and China Agriculture Research System of MOF and MARA (No. CARS-30-Z-02).

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Reply:We have deleted and changed the declaration in the Acknowledgments Section of my manuscript.

5. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

Reply:Our submission have contained all raw data required to replicate the results of study.

6. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

Reply:I accept your suggestion that all authors decide on a data sharing plan before acceptance.

7. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

Reply:I have ensured that I have an ORCID iD.

Reviewer #1:

1. The authors selected Wangjiazhuangmaotao No.2, Zhongnongjinhui, and Chunmei as three representative varieties with strong, moderate, and weak cold hardiness ranks to determine EI and BR. However, Table 3 showed that Zhongnongjinhui and Chunmei are both medium cold-hardiness (MH) variety. In the subsequent results, there was a difference in data of BR between Zhongnongjinhui and Chunmei, both of which were MH variety. Does this indicat that the identification of cold hardness through LTE investment of is not as precise? Please explain in detail.

Reply:The definition of medium cold-hardiness (MH) variety is relatively broad, which can also be divided into three levels: high, medium, and low, so there may be differences in the EI and BR of Zhongnongjinhui and Chunmei.

2. Minor points:Please review the manuscript for punctuation errors throughout the text, e.g. line 88 "... electrical leakage (EI)]".

Reply:We have checked and corrected the the manuscript for punctuation.

We have corrected line 88 "... electrical leakage (EI)]".

3. Minor points:Please review the manuscript for the consistency of font size, e.g. line 96 "A total of …", and line 164 "…hardiness…".

Reply:We have reviewed and corrected the manuscript for the consistency of font size.

4. Minor points:Please carefully check the reference format and ensure consistency, e.g. line 346 "Am. J. Enol. Vitic", and line 352 "Annals of Botany".

Reply:We have checked and corrected the reference format and ensure consistency,e.g. line 346 "Am. J. Enol. Vitic", and line 352 "Annals of Botany", as well as others.

Reviewer #2:

1. There are multiple formatting errors throughout the manuscript, such as the incorrect use of spaces in lines 34, 66, 74, 77, etc.

Reply:We have checked and corrected formatting errors formatting errors, such as the incorrect use of spaces in lines 34, 66, 74, 77, etc.

2. Line 80: "the LTE determination method involved cutting" should not be formatted in bold.

Reply:We have checked and corrected formatting errors formatting errors.

3.Fig 1: Graphing error, there is no point C in the diagram.

Reply:We have checked and corrected the annotation error for point C in Fig 1.

4.Line 140: “There” should be “there”.

Reply:“There” is correct writing, and we have corrected the punctuation error before it. 5. Line 256: An “of” should be removed.

Reply:We have checked and corrected the error.

5.Line 260: “wiht” should be “with”.

Reply:We have checked and corrected the error.

7. Line 278: “siince” should be “since”.

Reply:We have checked and corrected the error.

6.PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

Reply:Authors agrees to option to publish the peer review history.

Attachment Submitted filename: Response to Reviewers.docx

10.1371/journal.pone.0306914.r003
Decision Letter 1
Fu Guanfu Academic Editor
© 2024 Guanfu Fu
2024
Guanfu Fu
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
28 May 2024

PONE-D-23-42629R1Analyzing Cold Hardiness (Based on DTA) of One-Year-Old Branches of PeachesPLOS ONE

Dear Dr. Tian,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 12 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Guanfu Fu, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

According to the comments, many misspellings were still found in the manuscript. it could be accepted after careful checking.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #3: The revised manuscript provided by Li et al. has revised and respond correctly to comments from both former reviewers.

However, there is one more comment need to be amended. I found that the authors mentioned “Huangjinmi No.1” in the Abstract section of the manuscript, but I cannot find any related information about “Huangjinmi No.1” in the other sections of the manuscript. So please double check whether this is a typo of “Golden honey No. 1” (as I have read it in the Results section but not mentioned in the Abstract section) and amend the fault. I suggest the manuscript could be accepted after this fault being fixed.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

10.1371/journal.pone.0306914.r004
Author response to Decision Letter 1
Submission Version2
22 Jun 2024

Date: June 22, 2024

Dear Editors:

Thank you for your letter and for the academic editor and the reviewers’ comments concerning our manuscript “Analyzing Cold Hardiness (Based on DTA) of One-Year-Old Branches of Peaches”. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval.

Academic Editor

Journal Requirements:

Q:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

A:We have carefully checked the reference list to ensure it is complete and correct.

Responses to Editor：

Reviewers' comments:

Responses to Reviewers：I will no longer explain the affirmative response given by the reviewers, but I will provide my answers to the No and No responses given by the reviewers.

Q:5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #3: (No Response)

A:We have found an English native speaker with a research background to review our manuscript during revision. And if you think there is any problem, you can raise it at any time. we will look for professional organizations to improve the language

Q:6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #3: The revised manuscript provided by Li et al. has revised and respond correctly to comments from both former reviewers.

However, there is one more comment need to be amended. I found that the authors mentioned “Huangjinmi No.1” in the Abstract section of the manuscript, but I cannot find any related information about “Huangjinmi No.1” in the other sections of the manuscript. So please double check whether this is a typo of “Golden honey No. 1” (as I have read it in the Results section but not mentioned in the Abstract section) and amend the fault. I suggest the manuscript could be accepted after this fault being fixed.

A:About Golden honey No. 1 and Huangjinmi No.1 problem mentioned by the third reviewer, we have altered to use a Golden honey No. 1. (see abstract）

Q:While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at <a href="mailto:figures@plos.org">figures@plos.org. Please note that Supporting Information files do not need this step.

A:We have uploaded figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.

Attachment Submitted filename: Response to Reviewers.docx

10.1371/journal.pone.0306914.r005
Decision Letter 2
Fu Guanfu Academic Editor
© 2024 Guanfu Fu
2024
Guanfu Fu
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
26 Jun 2024

Analyzing Cold Hardiness (Based on DTA) of One-Year-Old Branches of Peaches

PONE-D-23-42629R2

Dear Dr. Qihang Tian

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Guanfu Fu, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

10.1371/journal.pone.0306914.r006
Acceptance letter
Fu Guanfu Academic Editor
© 2024 Guanfu Fu
2024
Guanfu Fu
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
28 Jun 2024

PONE-D-23-42629R2

PLOS ONE

Dear Dr. Tian,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

prof. Guanfu Fu

Academic Editor

PLOS ONE
==== Refs
References

1 Wu XQ , Mason AM , Yu ML , Ma RJ , Yu ZF . Quantitative proteomic analysis of pre- and post-harvest peach fruit ripening based on iTRAQ technique. Acta Physiol Plant. 2017; 39 : 181. doi: org/10.1007/s11738-017-2478-6
2 Li YH , Wang ZY , Tian QH , Zhou YC , Xu JT , et al . Quantitative proteomic analyses on the mechanisms of cold tolerance in two peach cultivars (Prunus persica L.Batsch) based on iTRAQ. European Jourticultural of Horticulture Science. 2021; 86 (3 ): 308–319. doi: org/10.17660/eJHS.2021/86.3.10
3 Niu RX , Zhao XM , Wang CB , Wang FL . Transcriptome profiling of Prunus persica branches reveals candidate genes potentially involved in freezing tolerance. Scientia Horticulture. 2020; 259 :108775. doi: org/10.1016/j.scienta.2019.108775
4 Ghasemi Soloklui A A , Ershadi A , Fallahi E . Evaluation of cold hardiness in seven Iranian commercial pomegranate (Punica granatum L.) cultivars. HortScience. 2012; 47 (12 ):1–5. doi: org/10.21273/HORTSCI.47.12.1821
5 Hyunsuk S , Youngjae O , Daeil K . Differences in cold hardiness, carbohydrates, dehydrins and related gene expressions under an experimental deacclimation and reacclimation in Prunus persica. Physiologia Plantarum. 2015; 154 (4 ): 485–499. doi: 10.1111/ppl.12293 25272204
6 Yu DJ , Hwang JY , Chung SW , Oh HD , Yun SK , et al . Changes in cold hardiness and carbohydrate content in peach (Prunus persica) trunk bark and wood tissues during cold acclimation and deacclimation. Scientia Horticulture. 2017; 219 : 45–52. doi: org/10.1016/j.scienta.2017.02.038
7 Ma YY , Huang DD , Chen CC , Zhu SH , Gao JG . Regulation of ascorbate-glutathione cycle in peaches via nitric oxide treatment during cold storage. Scientia Horticulture. 2019; 247 : 400–406. doi: org/10.1016/j.scienta.2018.12.039
8 Guy CL . Freezing tolerance of plants: current understanding and selected emerging concepts. Canadian Journal of Botany. 2003; 81 (12 ):1216–1223. doi: org/10.1139/b03-130
9 Repoa T , Mononenb K , Alvilab L , Pakkanenb TT , Hänninenc H . Cold acclimation of pedunculate oak (Quercus robur L.) at its northernmost distribution range. Environmental and Experimental Botany. 2008; 63 , 59–70. doi: org/10.1016/j.envexpbot.2007.10.023
10 Proebsting EL , Ahmedullah M , Brummund VP . Seasonal changes in low temperature resistance of grape buds. American Journal of Enology and Viticulture. 1980; 31 : 329–336. doi: 10.1007/BF01042416
11 Andrews PK , Sandidge CR , Toyama TK . Deep supercooling of dormant and deacclimating Vitis buds. American Journal of Enology and Viticulture. 1984; 35 : 175–177. doi: org/10.5344/ajev.1984.35.3.175
12 Jones KS , Paroschy J , McKersie BD , Bowley SR . Carbohydrate composition and freezing tolerance of canes and buds in Vitis vinifera. Journal of Plant Physiology. 1999; 155 : 101–106. doi: org/10.1016/s0176-1617 (99)80146-1
13 Ferguson JC , Tarara JM , Mills LJ , Grove GG , Keller M . Dynamic thermal time model of cold hardiness for dormant grapevine buds. Annals of Botany. 2011; 107 (3 ): 389–396. doi: 10.1093/aob/mcq263 21212090
14 Gu SL . Lethal temperature coefficient-a new parameter for interpretation of cold hardiness. Journal of Horticultural Science and Biotechnology. 1999; 74 (1 ): 53–59. doi: 10.1080/14620316.1999.11511071
15 Yoshida SZ , Sakai A . Phospholipid changes associated with the cold hardiness of cortical cells from poplar stem1. Plant and cell physiology. 1973;14 (2 ): 353–359. doi: org/10.1093/oxfordjournals.pcp.a074867
16 Burke MJ , Gusta LV , Quamme HA . Weiser CJ, Li PH. Freezing and injury in plants. Annual Review Plant Biology. 1976; 27 : 507–528. doi: org/10.1146/annurev.pp.27.060176.002451
17 Cline JA , Beneff A , Edwards M . Cold hardiness of select apple cider cultivars in Canada. Canadian Journal of Plant Science. 2022; 102 (2 ): 394–404. doi: org/10.1139/cjps-2021-0167
18 Quamme HA , Chen PM , Gusta LV . Relationship of deep supercooling and dehydration resistance to freezing injury in dormant stem tissue of ‘Starimson Delicious’ apple and ‘Siberian C’ peach. Journal of the American Society for Horticulture Science. 1982; 107 (2 ):299–304.
19 Minas LS , Sterle D . Differential thermal analysis sheds light on the effect of environment and cultivar in peach floral bud cold hardiness. Acta horticulturae. 2020; 385–392. doi: 10.17660/ActaHortic.2020.1281.51
20 Quamme HA , Stushnoff C , Weiser CJ . The relationship of exotherms to cold injury in apple stem tissues. Journal of the American Society for Horticulture Science. 1972; 97 (5 ): 608–613. doi: 10.21273/JASHS.97.5.608
21 Mills LJ , Ferguson JC , Markus Keller M . Cold-hardiness evaluation of grapevine buds and cane tissues. American Journal of Enology and Viticulture. 2006; 57 (2 ): 194–200. doi: 10.1016/j.scienta.2005.07.007
22 Yun SK , Bae H , Chung K-H , Yoon IK , Nam EY , et al . Sugar, starch, and proline in peach trees exposed to freezing temperatures during dehardening. Agriculture Sciences. 2014; 5 : 913–921. doi: org/10.4236/as.2014.510099
23 Gao Z , Li J , Zhu HP , Sun LL , Du YP , Zhai H . Using differential thermal analysis to analyze cold hardiness in the roots of grape varieties. Scientia Horticulturae. 2014; 174 : 155–163. doi: org/10.1016/j.scienta.2014.05.002
24 Levitt J. The hardiness of plants. New York, London: Academic Press; 1956.
25 Guo Y , Li Y , Zhang SH , Zhang XF , Wang GP . Using low temperature exotherms method to analyze branch cold resistance of chinese chestnut. Acta Agriculturae Boreali-sinica 2017; 32 : 201–209. (in Chinese) doi: 10.7668/hbnxb.2017.S1.035
