Comparison of Cold Tolerance in Three Ground-cover Chrysanthemum Cultivars during Wintering in the Open Field

Xin ZHOU Man SHEN Pingsheng LENG

Abstract    [Objectives]  Among many chrysanthemum types， the ground-cover chrysanthemum cultivar group has the characteristics of dwarf plant， long flowering period， rich color and strong resistance to stress. This study aimed to screen out cultivars with strong cold tolerance in order that they can be used for planting in urban green space and economic forests in northern cold areas， so as to enrich the plant community structures of landscape in cold regions.

[Methods] Three ground-cover chrysanthemum cultivars，  i.e. ， ‘Meiaihuang’， ‘Xinhong’ and ‘Zixunzhang’， were selected as the experimental materials. During the wintering period in the field in Beijing， they were observed and measured for the changes of the morphological manifestations and the physiological indices， and the relationship between the indices and low temperature was analyzed. The cold tolerance of the three ground-cover chrysanthemum cultivars was also comprehensively evaluated.

[Results]  During the open-field overwintering period， the root activity of the three ground-cover chrysanthemum cultivars increased slowly with cooling， decreased at the freezing low temperature， and then increased again in early spring. With natural cooling， the membrane injury percentages （MIPs） of the leaves and foot shoots increased significantly， and the contents of malondialdehyde （MDA） increased; the contents of soluble sugar （SS） and proline （Pro） gradually accumulated， and the contents of SS and Pro in foot shoots increased significantly in late winter; and the superoxide dismutase （SOD） activity showed a similar trend of rising slowly at first and then increasing significantly. There were correlations between MIPs in the leaves and foot shoots of the three cultivars and other physiological indices： a significant negative correlation with MDA content， and a significant positive correlation with SS content， Pro content and SOD activity.

[Conclusions]  Combining the morphological manifestations and physiological indices， the cold tolerance of the three ground-cover chrysanthemum cultivars was evaluated by the subordinate function method. The results showed that the cold tolerance of the three ground-cover chrysanthemum cultivars ranked as ‘Meiaihuang’>‘Xinhong’>‘Zixunzhang’. The strong cold tolerance of foot shoots and roots was the main reason that the three ground-cover chrysanthemum cultivars could overwinter in the open field.

Key words   Ground-cover chrysanthemum; Cold tolerance; Overwinter in the open field; Comprehensive evaluation

Chrysanthemum （ Chrysanthemum morifolium ） is a perennial herbaceous flower of  Chrysanthemum  in Compositae. It has a long history of planting in China and is of high ornamental value.  Among  many varieties of the chrysanthemum family， ground-cover chrysanthemum （ Dendranthema×grandiflorum  Kitamura）， a new cultivar group， has the characteristics of dwarf plants， long flowering period， compact flower pattern， strong stress resistance and extensive cultivation resistance， and can be used as an excellent ground-cover plant. With the increasing demand for autumn flowering plant species in urban landscape greening， the ground-cover chrysanthemum cultivars that can overwinter in the field in the  "Three  North" areas will play an important role in urban greening and beautification.

Low temperature is one of the main factors limiting the survival and distribution of organisms， affecting the growth and development of plants. Under low temperature stress， plants will produce a series of ecological adaptive responses， including various aspects of morphological structure， physiological and biochemical processes and gene expression. Plant cold resistance （cold tolerance） is a hereditary expression affected by its physiological and biochemical characteristics， and their cold resistance mechanism is intricate  [1-2] .

If ground-cover chrysanthemum is applied to urban green space and economic forests in the northern cold region for mixed planting， the primary consideration will be whether the plants cultivated in the field can safely survive winter.

Based on the above research background and purpose， in this study， three ground-cover chrysanthemum cultivars ‘Meiaihuang’， ‘Xinhong’ and ‘Zixunzhang’ were selected as materials and observed for the changes in morphological performance and physiological indicators during overwintering in the field in Beijing， and a comprehensive evaluation of the cold tolerance in the three cultivars was conducted， in order to study the physiological characteristics of cold tolerance of ground-cover chrysanthemum and establish an  evaluation  index system for screening cold-tolerant ground-cover chrysanthemum cultivars and selecting cultivars with strong cold tolerance for use in landscaping in northern cold regions.

Materials and Methods 

Experimental materials

Three ground-cover chrysanthemum cultivars ‘Meiaihuang’， ‘Xinhong’ and ‘Zixunzhang’ were used as plant materials. On May 1， 2018， they were planted in the horticultural experimental site of Beijing University of Agriculture with a plant-row spacing of 80 cm×60 cm， and normal field water and fertilizer management was adopted.

Experimental methods

During the overwintering period from October 2020 to March 2021， the foot shoots and leaves were sampled for 15 d/time， and the root system was sampled for 30 d/time. Biological sampling and photography were performed on the three ground-cover chrysanthemum cultivars cultivated in the field.

According to the weather forecast， the weather conditions were recorded. Every day at 8：00 am and 2：00 pm， a hand-held digital thermometer （Shanghai Yueqiu Instrument Co.， Ltd.） was used to measure the surface temperature of the test site and the soil temperature at a depth of 20 cm from the surface  [3]  to obtain the daily average temperature and daily minimum temperature， and then obtain the monthly average maximum temperature and monthly average minimum temperature.

Observation of plant morphology

From October 2020 to March 2021， the morphological indices of the test plants were recorded and photographed.

Determination of physiological indicators

At 10：00 am， leaf， foot shoot and root samples were collected for the determination of physiological and biochemical indices. Various physiological indices were carried out according to the methods of Li  [4] ， Hao   et al.    [5]  and Li  et al.   [6] . Specifically， the membrane injury percentage （MIP） was measured by the conductivity method; the content of MDA was determined by the thiobarbituric acid （TBA） method; the content of proline was determined by acidic ninhydrin colorimetry; the content of soluble sugar was determined by anthrone colorimetry; the content of soluble protein was determined by the coomassie brilliant blue method; SOD activity was measured by the NBT photoreduction method; and root activity was measured by the triphenyltetrazolium chloride （TTC） method.

Data statistics and analysis

The experimental data were analyzed and plotted using  Microsoft  Excel 2010 and SPPS 22 software. The membership function method was used to comprehensively evaluate the cold  tolerance.

Results and Analysis 

Variation of ambient temperature in the experimental site during overwintering in the field

During the natural cooling process of overwintering in the field， accurately grasping the dynamic changes of the ambient temperature of experimental sites not only helps to compare and analyze the differences in the cold tolerance of different cultivars of ground-cover chrysanthemum， but also helps to quantify the trend of temperature changes and understand the threshold of temperature stress  [7-8] . In this study， the monthly average temperatures of the atmosphere， earth surface and soil were continuously measured and recorded， from September 2020 to April 2021 （Fig. 1）. It can be seen from Fig. 1 that as the temperature gradually decreased， from December 2020 to January 2021， the monthly lowest temperature of atmosphere （earth surface） and soil in the field were （-5±1） ℃.

Morphological changes of leaves， foot shoots and roots of the three ground-cover chrysanthemum cultivars during overwintering in the field

Fig. 2 shows the morphological changes of leaves， foot shoots and roots of the three ground-cover chrysanthemum cultivars during the overwintering period in the field from October 2020 to March 2021.

Fig. 2 shows that on October 30， 2020， the three ground-cover chrysanthemum cultivars were in the final flowering stage， and the climate entered the early frost stage. Although the leaves of the plants had fallen frost thereon， they were not completely withered， and some of the leaves were dark green. On November 30， 2020， it could be observed that most of the leaves of the three   ground-cover chrysanthemum cultivars had withered， and the leaf color had turned dark brown. The foot shoots that sprouted at the root and neck parts were still growing in mid-to-late November. In late December， it was observed that some foot shoots remained green after snow， but as the subzero low temperature continued until late January 2021， most of the foot shoots at the root and neck parts withered and wilted until they germinated in early spring in March 2021 again. The root system remained active throughout the winter， and although the lateral roots of the root system were observed to be black and die in January and February 2021， most of the main roots remained white or light brown.

From the observation results of the leaves， foot shoots and root system of the three ground-cover chrysanthemum cultivars during the whole overwintering process in the open field， all three cultivars could safely overwinter in the open field， and their cold tolerance seemed to be mainly determined by the vitality of the root system.

The cold tolerance of plants is an overall evaluation index  [9] . In order to further explore the cold tolerance performance of  the three  ground-cover chrysanthemum cultivars during overwintering in the open field， the measurement results of physiological  indices such as root activity were analyzed， in order to explore the

differences in the cold tolerance between different cultivars， and screen physiological indices of cold tolerance closely related to low  temperature.

Changes of physiological indices related to cold tolerance of the three ground-cover chrysanthemum cultivars during overwintering in the open field 

Root activity

Perennial flowers mainly rely on the vitality of their roots to survive in the open field in winter. Therefore， the root vitality status of plants can reflect the cold resistance of plants to a certain extent  [10] . It can be seen from Fig. 3 that as the ambient temperature decreased， the root activity values of ‘Xinhong’ and ‘Zixunzhang’ dropped to the lowest values on January 15， 2021， and the data of the two were close， while that of  ‘Meiaihuang’  reached the lowest value in early February， and the root activity was still close to 70 μg/（g·h）， which was about 50% higher than those of ‘Xinhong’ and ‘Zixunzhang’. The root activity of the three cultivars showed an upward trend in mid- February.

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Membrane injury percentage （MIP）

The phenomenon of increased plant cell membrane permeability under low temperature stress has been confirmed in many cold resistance research reports， and some researchers believe that the cell membrane permeability under low temperature stress directly reflects the degree of damage and cold resistance of plants  [11] . The higher the electrical conductivity of plants， the stronger the cell membrane permeability， the greater the damage to the cell membrane， and the lower the cold resistance  [12] .

In this study， the membrane injury percentages （MIPs） of leaves and foot shoots of the three ground-cover chrysanthemum cultivars were determined. It was found that with the obvious cooling in winter， the leaf MIP values of the three cultivars gradually increased （Fig. 4a）. Compared with September， the MIP values in early October did not change significantly （ P >0.05）， while from early to mid-November， the leaf MIP values of the three cultivars all reached the highest values， and there were no significant differences between cultivars （ P >0.05）.

The changes in the MIP values of the foot shoots of the ground-cover chrysanthemum cultivars with decreasing air temperature were similar to those of leaves （Fig. 4b）. From October to November 2020， the MIP values of the foot shoots of the three  cultivars  increased significantly for the first time， and the MIP  value  of the foot shoots of ‘Meiaihuang’ had the lowest increase （ P <0.05）. It was found from sampling and testing on January 1， 2021 that the MIP values of the foot shoots of the three cultivars increased for the second time. The MIP values of the foot shoots of the three cultivars were 70.48%， 84.35% and 80.67%， respectively， indicating that under subzero freezing， the membrane permeability of the foot shoots of the three cultivars increased， and the low temperature damage was greater.

Contents of osmotic adjustment substances

Osmotic adjustment substances are a class of protective substances. When plants are damaged by various stresses， the contents of osmotic adjustment substances in plants will change significantly  [13] .

As shown in Fig. 5a and Fig. 5b， with the decrease of the ambient temperature in the open field， the soluble sugar contents of the leaves and foot shoots of the three cultivars increased significantly in early November， and there were significant differences between the three varieties （ P <0.05）. Specifically， the soluble sugar content of ‘Meiaihuang’ was  134.71  mg/g， which was  16.08%  higher than that of ‘Xinhong’ and 56.86% higher than that of ‘Zixunzhang’. In mid-November， the soluble sugar contents of the leaves and foot shoots of ‘Meiaihuang’ and ‘Zixunzhang’ increased， and there were significant differences between ‘Meiaihuang’ and other two cultivars （ P <0.05）. With the decrease of temperature， the peaks of soluble sugar content in the foot shoots of the three cultivars all appeared in late December. The soluble sugar content in the foot shoots of ‘Xinhong’ was  303.30  mg/g， and the soluble sugar content in the foot shoots of  ‘Zixunzhang’  was 249.42 mg/g， which was the lowest among the three cultivars. In the process of overwintering in the open field， the soluble sugar content of ‘Meiaihuang’ was higher than those of ‘Xinhong’ and ‘Zixunzhang’ regardless of whether it came  from leaves or foot shoots; and with the decrease of ambient temperature， the differences from the two were significant （ P <0.05）.

Meanwhile， it can be seen from Fig. 5c that the change trends of proline content in the leaves of the three ground-cover chrysanthemum cultivars during overwintering was roughly similar to those of soluble sugar content. The peaks appeared on November 1， and the leaf proline content of ‘Meiaihuang’ was the highest， followed by ‘Xinhong’， and ‘Zixunzhang’ which was the lowest. The proline content in the foot shoots of ‘Meiaihuang’ was significantly different from those of ‘Xinhong’ and ‘Zixunzhang’ （ P <0.05）， and reached the highest value in mid-to-late December （Fig. 5d）.

During the overwintering process， the contents of soluble sugar and proline in the three ground-cover chrysanthemum cultivars were relatively stable from October to November （the monthly average temperature was about 5-6 ℃. When the ambient temperature continued to drop and exceeded the temperature threshold that the plant itself can withstand （close to 0 ℃）， the protective substances inside the plant cells rapidly accumulated in large quantities to resist low temperature adversity  [14] .

MDA content and SOD activity

The cell membrane is the primary part of plants damaged by low temperature. With a series of signal transduction in plants under low temperature， the cell membrane will undergo oxidation reaction and produce oxidation products， thus affecting the normal growth of plants  [15-16] .

The content of MDA is the product of membrane lipid peroxidation， and the change of its content reflects the degree of plant damage and is one of the important signs of damage to plant membranes under stress  [17] . Therefore， the determination of MDA content is an effective method to determine the cold resistance of plants.

As shown in Fig. 6， the changes of MDA content in the leaves of the three ground-cover chrysanthemum cultivars from October to November showed an upward trend. Compared with the MDA content of leaves sampled on October 15 among the three cultivars， ‘Zixunzhang’ had the highest MDA content and the largest increase （3.5 times）， while ‘Meiaihuang’ showed the smallest increase of MDA content in the leaves （1.4 times）. It showed that when the temperature dropped below 5 ℃ （Fig. 1）， ‘Zixunzhang’ had the greatest stress response to cooling. In mid-November， the contents of MDA in leaves of the three cultivars all reached their highest values.

The MDA contents in the foot shoots of the three cultivars also increased significantly on October 15， but there were no significant changes in the subsequent late autumn （Fig. 6b）. A gradual upward trend of accumulation occurred from mid-November to January of the following year when the temperature dropped below 0 ℃  （Fig. 1） ， and at the beginning of January， the MDA contents of the foot shoots of the three cultivars all reached their peaks. At this time， the content of MDA in the foot shoots of ‘Meiaihuang’ was the lowest among the three， showing significant differences. Specifically， it was 11.96% lower than that of ‘Xinhong’ and 22.12% lower than that of ‘Zixunzhang’， indicating that ‘Meiaihuang’ had better cold tolerance than the two.

SOD is one of the most important free radical-scavenging enzymes in plant cells. It plays an important role in the defense of reactive oxygen species in aerobic organisms， and it can specifically scavenge superoxide anion free radicals and relieve stress damage  [18] .

Fig. 7a shows that the SOD activity in the leaves of ‘Meiaihuang’， ‘Xinhong’ and ‘Zixunzhang’ did not change significantly in early October， but increased significantly in November， with an increase of 72.75%， 77.36% and 89.24%， respectively， which showed that the self-defense mechanism of the three cultivars against cold temperatures had been activated at this time. By mid-November， as the temperature continued to drop， the SOD activity of the leaves of the three cultivars decreased significantly， and the morphological observation records from the three cultivars also showed that the leaves of the plants had been irreversibly damaged at this time.

The changes of SOD activity in the foot shoots of the three cultivars during overwintering in the open field are shown in Fig. 6b. During mid-October， when the temperature dropped below 11-13 ℃ （Fig. 1）， the SOD activity decreased significantly. From  November， when the temperature dropped to about 5-6 ℃ （Fig. 1），  the SOD activity of ‘Meiaihuang’， ‘Xinhong’ and ‘Zixunzhang’ reached 318.91， 288.00 and 259.26 U/（g·min）， respectively， indicating that the low temperature at this time had an impact on the foot shoots of ground-cover chrysanthemum， which caused the SOD enzyme in its body to maintain a high activity to produce a protective effect. In late winter， the SOD activity in the foot shoots of ‘Xinhong’ and ‘Zixunzhang’ decreased slightly， while the SOD activity in the foot shoots of ‘Meiaihuang’ was still the highest among the three cultivars， and the differences were significant between the three （ P <0.05）.

Evaluation of cold tolerance of the three ground-cover chrysanthemum cultivars

Comprehensively evaluating the physiological characteristics and quality characteristics of plants adapting to adversity by the membership function analysis is one of the scientific methods  adopted  by researchers and has been widely used in the comprehensive  evaluation  of various plants  [19-20] .

In this study， correlation analysis was also carried out on the physiological indices measured on the leaves and foot shoots of the three ground-cover chrysanthemum cultivars during the natural cooling process （Table 1）， and the membership function method was applied to evaluate the cold tolerance of the three cultivars.

It can be seen from Table 1 that the MIPs of the leaves and foot shoots were significantly negatively correlated with their MDA contents （ P <0.01）， and the correlation coefficients  R  were  -0.996  and -0.994， respectively. There was an extremely significant positive correlation between MIPs in leaves and foot shoots with their corresponding proline content and SOD activity （ P <0.01）. The degree of the correlation between MIP and soluble sugar content was related to plant parts. The MIP in the foot shoots had an extremely significant positive correlation with soluble sugar content （ R =0.967，  P <0.01）， and the MIP in the leaves had a significant positive correlation with soluble sugar content （ R =0.830，  P <0.05）.

After the correlation analysis between the five physiological indices in the leaves and foot shoots of the ground-cover chrysanthemum plants， the overall cold tolerance of the three cultivars in the deep winter period（sampled on December 30， 2020）was further evaluated comprehensively （Table 2）.

Through the membership function method， the cold tolerance of the three ground-cover chrysanthemum cultivars was sorted， and the results showed an order of ‘Meiaihuang’ > ‘Xinhong’ > ‘Zixunzhang’.

Conclusions and Discussion

During the wintering of plants in the open field， in addition to measuring the relevant physiological indices to judge their cold tolerance， it is also necessary to observe the growth status and morphological performance of the plants. Wan  et al.   [21] ， Li   et al.    [22]  and Wang  et al.   [23]  identified the cold tolerance of a variety of evergreen broad-leaved plants and 58 deciduous woody plants， peach， based on the observation of the wintering performance of these tree species. However， during overwintering， the changes in the external morphology of plants can only show the response of plants to low temperature to a certain extent， and the cold tolerance of plants cannot be quantified. Therefore， it is necessary to combine the measurement of physiological indices to make a comprehensive judgment.

The cell membrane is the barrier between plants and the natural environment， and the impact of low temperature stress on plants first acts on the plasma membrane. When plants are injured by low temperature， membrane lipid peroxidation occurs in the cell membrane system， and the accumulated final product MDA damages the cell membrane and causes electrolyte extravasation. Therefore， cell membrane permeability （membrane injury percentage， MIP） and MDA content are important physiological indices that reflect the degree of cell membrane injury and measure the cold tolerance of plants  [24-25] . After plants are injured by low temperature， the lower the contents of MIP and MDA， the stronger the cold tolerance of plants. Otherwise， the cold tolerance is weaker  [16] . The results of this study found that the MIP and MDA contents in the leaves and foot shoots of ‘Meiaihuang’ were lower than those of ‘Xinhong’ and ‘Zixunzhang’ during the open-field overwintering period of the three ground-cover chrysanthemum cultivars， and the contents of MIP and MDA in the leaves and foot shoots of ‘Meiaihuang’ were lower than those of ‘Xinhong’ and ‘Zixunzhang’， and in the deep winter cold season from November to January， it showed stronger cold tolerance.

Changes in plant root function will inevitably affect the physiological characteristics of above-ground leaves  [26] . Root activity will change under low temperature stress conditions. In this study， it was found that， the root activity of the three ground-cover chrysanthemum cultivars declined slowly during the slow cooling process in the open field in early October 2020， and showed a downward trend in January 2021 when the temperature dropped to the lowest temperature below zero， while when the temperature warmed up in the late winter， the root activity increased. It shows that a certain threshold of low temperature will lead to the decline of root activity， but the process of slow cooling in nature may make the roots of plants cultivated in the open field have a certain degree of low temperature adaptation （exercise）， and after being acclimated by low temperature within a certain low temperature range， the plant can withstand low temperature damage and maintain the stability of root viability. Therefore， even if the aboveground part is frozen and died， due to the survival of the root system， the normal germination of the aboveground part can be guaranteed in the following spring.

It may also be the reason why the cold tolerance observed in the natural cooling process of plants cultivated in the open field is higher than that measured under artificial low temperature stress treatments in the laboratory. Because laboratory conditions ignore the differences in the temperature of various parts of plants （aboveground and underground parts， air temperature and soil temperature） and their inherent cold tolerance （leaves， foot shoots and roots）， field plants have a survival mechanism to  avoid  freezing during overwintering in the open field  [27] .

The cold resistance of ground-cover chrysanthemum is a genetic manifestation affected by the comprehensive effects of its physiological and biochemical characteristics， and its cold resistance mechanism will be complicated  [28] . Judging and measuring the cold tolerance of ground-cover chrysanthemum based only on a single physiological index is inevitably biased. Therefore， it is of great practical significance to identify and evaluate the cold tolerance of ground-cover chrysanthemum by combining multiple physiological indices with mathematical analysis methods.

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