South African Journal of Botany 94 (2014) 140–148

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South African Journal of Botany

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Physiological and biochemical responses of six herbaceous peonycultivars to cold stress

Qi Wang a,b, Tangren Cheng c, Xiaonan Yu a,c,⁎, Jaime A. Teixeira da Silva d,⁎⁎, David H. Byrne e

a College of Landscape Architecture, Beijing Forestry University, No. 35 Qinghuadong Road, Beijing 100083, PR Chinab School of Architecture, Tianjin Chengjian University, No. 26 Jinjing Road, Tianjin 300384, PR Chinac Beijing Key Laboratory of Ornamental Plants Germplasm Innovation andMolecular Breeding, National Engineering Research Center for Floriculture, No. 35QinghuadongRoad, Beijing 100083, PRChinad P. O. Box 7, Miki cho Post Office, Ikenobe 3011-2, Kagawa-Ken 761-0799, Japane Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843-2133, USA

⁎ Correspondence to: X. Yu, College of LandscapeUniversity, No. 35 Qinghuadong Road, Beijing 100083, PR⁎⁎ Corresponding author. Tel.: +81 878988909.

E-mail addresses: yuxiaonan626@126.com (X. Yu), jai(J.A. Teixeira da Silva).

http://dx.doi.org/10.1016/j.sajb.2014.05.0120254-6299/© 2014 SAAB. Published by Elsevier B.V. All ri

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 August 2013Received in revised form 10 March 2014Accepted 13 May 2014Available online

Edited by JM Farrant

Keywords:Relative electric conductivitySoluble sugarSoluble proteinMalondialdehydeProlineSuperoxide dismutase

Although herbaceous peony cultivars have been introduced and promoted widely, a key limitation to their use istheir level of cold hardiness. In this study, the physiological and biochemical responses of six herbaceous peony cul-tivars to different temperatures and periods of exposure to freezing temperatures were assessed by analyzingchanges in six indices. Under our experimental conditions, relative electric conductivity, soluble sugar concentration,soluble protein concentration, and malondialdehyde concentration generally increased as temperature declined,while proline content decreased. Superoxide dismutase activity fluctuated. After the same indicators were used toassess cold resistance over time at−20 °C, the six cultivars could be ranked, in decreasing levels of cold resistance,as: ‘Da Fu Gui’, ‘Fen Yu Nu’, ‘Kansas’, ‘Monsieur Jules Elie’, ‘Taff’, and ‘Pink Hawaiian Coral’.

© 2014 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction

Due to the sessile nature of plants, theymay be subjected to injury byextreme environmental conditions. Plant hardiness is a genetic character-istic that originates from the long-term adaptation to low temperatureenvironmental changes (Chen and Liu, 2003). The expression of plantcold-resistance genes is closely related to environmental conditions andplant physiological activities (Sakai, 1987; Hughes and Dunn, 1996;Thomashow, 1999). Conversely,manyphysiological andbiochemical pro-cesses can reflect a plant's response to environmental stresses (Lamberset al., 1998). An understanding of the physiological and biochemicalmechanisms of cold hardiness is a prerequisite for efficient breeding aswell as the introduction and management of plants into cold or freezingenvironments.

The peony, a perennial herb, has a cultivation history of more than3900 years (Li, 1999; Shen et al., 2012) and is currently a sought-aftercut flower in international markets. Peony cultivars used for cut flowers

Architecture, Beijing ForestryChina. Tel.: +86 15810050531.

metex@yahoo.com

ghts reserved.

are divided into three groups: Lactiflora, Hybrid or Intersectional group(Qin, 2004; Yao, 2009). Most herbaceous peony cultivars are in theLactiflora group which is derived from the Chinese species Paeonialactiflora Pallas (Kamenetsky et al., 2003). More recently the focus ofmuch herbaceous peony breeding has been shifting to the Hybridgroup which is a cultivar series formed by a plurality of species withmany advantages such as unique flower patterns and bright saturatedcolors. The six herbaceous peony cultivars used in this study are fromthe Lactiflora and Hybrid groups. As with many other perennial plantsin temperate climates, herbaceous peonies have a perennial crown(metamorphosed underground shoot) which serves to accumulate stor-age products for plant renewal in the new growth season (Kapinos andDubrov, 1993; Kamenetsky et al., 2003).

Although there has been some research on the physiological andbiochemical responses of herbaceous peonies to heat stress (Lü andLiu, 2008), heat and humidity tolerance (Liu, 2008) and soil droughtstress (Guo, 2009), there has been little research on cold stress. In con-trast, tree peonies have been evaluated for their relative cold hardinessin northern China (Inner Mongolia and Harbin). This work showeddifferences among species (Paeonia rockii T. Hong et J.J. Li, Paeoniasuffruticosa Andr.) and cultivars within species (Li, 2009; Ren, 2009;Zhao et al., 2011). Ju (2011), studying the ecological adaptability andcold resistance of 12 tree peony cultivars in Northeast China, found

141Q. Wang et al. / South African Journal of Botany 94 (2014) 140–148

that proline concentration and soluble polysaccharide concentration inshoots during winter as well as leaves during the growing seasoncould be used to assess tree peony cold hardiness.

Membrane stability is closely related to plant cold hardiness (Peter,1984). As early as 1912, Maximov (1912) suggested that disruption ofthe plasma membrane was the primary cause of freezing injury. In1932, Dexter et al. (1932) developed a conductivity method for measur-ing cold hardiness of plants which has long been used as one of themain methods for identification of plant cold resistance. Undergroundbuds and roots of herbaceous peonies are the only two remaining organsin the winter. However, there are more errors in the method of relativeelectric conductivity (REC) for root measurement (Li et al., 1993). In thisexperiment, the underground buds were used to measure REC becausethese organs are less affected by the thickness of slices and other factors,and the roots were used to measure other indices since they are easilyobtained and closely related to the growth of herbaceous peonies in thenext year. The basic assumption of this method is that the greater theinjury of the living tissue, the greater the efflux of ions from the thawedcells (Jiwan et al., 1977). Freezing tolerance is a result of several cryopro-tectivemechanisms operating concurrently (Sakai, 1987). Because com-patible solutes accumulate during cold acclimation, it is thought that thisaccumulation is a cryoprotective mechanism in some plants (Alberdiand Corcuera, 1991; Livingston, 1996). Soluble carbohydrates, proteinand free proline may be involved in freezing point depression of cellsap and prevention of plasmolysis during cell dehydration caused byfreezing (Sakai, 1987; Santarius, 1992). A cryoprotective function of pro-line, which accumulates in response to osmotic stresses such as drought,salt and cold in many plant species, has been demonstrated (Bajguz,2009). The accumulation of malondialdehyde (MDA), which acts as anend product of lipid peroxidation, is considered to reflect the physiolog-ical state of plant membrane lipids under cold stress (Imahori et al.,2008). Superoxide dismutase (SOD) and peroxidase (POD) play adefinitive role in low-temperature stress (Li et al., 2000; Lin et al.,2005; Xu et al., 2011; Mansour et al., 2012). SOD and POD enhancethe protection of membrane structures, through increased activity,and are thus considered to be the main anti-oxidizing agents in plants.SOD is a metalloprotein catalyzing the dismutation of the superoxidefree radical (O2

•−) to molecular oxygen and H2O2 (Constantine andStanley, 1977; Cervilla et al., 2007).

The objective of this study was to determine how changing temper-atures and the length of exposure to low (freezing or below-freezing)temperatures affect the physiology and biochemistry of six herbaceouspeonies. Using these indices and the relative cold hardiness, a theoreti-cal basis was established for the future introduction of these cultivarsinto cold environments.

2. Materials and methods

2.1. Plant material and experimental treatments

The six herbaceous peony cultivars (Table 1) were collected fromnurseries in Jiufeng and Xiaotangshan, Beijing, all of which had beendivided three years previously.

Table 1Characteristics of the six herbaceous peony cultivars used in this study.

Cultivar Location developed Groupa Flower colo

‘Da Fu Gui’ China Lactiflora Red‘Fen Yu Nu’ China Lactiflora Pinkish pur‘Monsieur Jules Elie’ France Lactiflora Pink‘Kansas’ U.S.A. Lactiflora Deep red‘Taff’ U.S.A. Lactiflora Pink‘Pink Hawaiian Coral’ U.S.A. Hybrid Coral pink

Note: Information based on Yu et al. (2011a) and http://www.paeo.de (Carsten Burkhardt's Wa Lactiflora — intercultivar hybrid, hybrid— interspecific hybrid.b S — single, SD — semi-double, D — double.

On October 10th, 2011, the dormant peonies were pruned to theground, the roots lifted out of the ground and washed, and then witha sharp sterilized knife cut into divisions containing five to eight strongbuds and fleshy root. The roots were shortened to 15 to 20 cm and thesmaller, unhealthy roots were removed.

Fifteen plants of each cultivar that had been divided as indicatedabove andwith root diameters between 1.2 cmand 1.8 cmwere plantedwith the samemass of substrate (peat:perlite:vermiculite= 3:1:1, v/v)in the same plot of a nursery in Xiaotangshan, Beijing. Pit depth wasbetween 25 and 30 cm. Plants were exposed to natural cold conditionsuntil mid-December (average temperature was approximately 0 °C).From the initial 15 plants, 8 were randomly selected and dug outcarefully on December 12th. From each, three underground budswere cut from the base. In addition, a 5-cm root (measuring fromthe root tip backwards) was cut, ensuring that the maximum diame-ter of each root was 1.5 cm. Three roots were removed from eachplant so that 24 buds and roots were obtained per cultivar. All experi-mental materials were wrapped separately in aluminum foil and takento the lab immediately for analyses. In the laboratory, the undergroundbuds and roots were washed thoroughly with running tap water, rinsedthree times with deionized water, then blotted dry on filter paper.Twenty-one buds and roots were selected randomly from the above 24buds and roots, and the experimental materials were divided into sevengroups (so that there were three buds and three roots in each part) andplaced in separate sealed plastic bags for the different low temperaturetreatments.

A programmed cooling device (Model: IceCube 14S; SY-LABCorp., Neupurkersdorf, Austria) was used to drop the temperaturein a controlled manner. All experimental materials were placed intothe device when its internal temperature had dropped from room tem-perature to 4 °C. Thereafter, the temperature was dropped at a rate of0.02 °C·min−1 to 0 °C. After 5 h, one sample of each cultivar was re-moved. The same protocol was applied every 5 h for the following tem-peratures: 0,−6,−12,−18,−24,−30 and−36 °C. The undergroundbuds of each cultivar that had been removed from the device wereplaced in a refrigerator at 4 °C and thawed for 8 h, and then the threeunderground budswere chopped andmixed evenly. The correspondingexperimentalmaterialswereweighed equally to determineREC. In con-trast, the three roots of each cultivarwere stored in liquid nitrogen priorto determination of physiological indices (soluble sugar concentration,soluble protein concentration, proline concentration, MDA concentra-tion and SOD activity). Before determining the indices, the roots werealso chopped, mixed and weighed as described for the undergroundbuds.

For the second experiment, five plants of each cultivar were random-ly chosen from the remaining stock plants and the same method wasused to collect underground buds and roots. This set of experimentalma-terials was divided into four parts for each cultivar such that 24 sampleswere placed into the programmed cooling device at 4 °C. Temperaturewas dropped at a rate of 0.02 °C·min−1. When −8 °C was reached,this temperature was maintained for 5 h, and then dropped at0.02 °C·min−1 to −20 °C. After 5, 15, 25 and 35 h at this tempera-ture, one sample (containing underground buds and roots) of each

r Flower diameter (cm) Flower typeb Plant height (cm)

15 D 85ple 12 S 68

13 D 8613 D 8612 D 7512 SD 90

eb Project Paeonia, n.d.).

142 Q. Wang et al. / South African Journal of Botany 94 (2014) 140–148

cultivar was removed and the experimental materials were handled asindicated above for determination of physiological indices. All the aboveexperiments were repeated three times.

2.2. Measurement methods of indices

The thawed underground buds were taken out from the refrigeratorand stripped of the outermost layer of scales and the appendages tothe inner part. The isolated bud was dried with filter papers and thenchopped up. Then the relative electric conductivity (REC) of electrolyteleakage was determined using the methods of Dexter et al. (1932) andLi (2000).

The anthrone method was used to determine soluble sugar concen-tration (Morris, 1948; Li, 2000), while the soluble protein concentrationwas prepared using the Coomassie Brilliant Blue G-250 staining method(Bradford, 1976). Proline concentration was determined using themethods of Troll and Lindsley (1955) and Li (2000), and the MDAconcentration was determined based on the methods of Heath andPacker (1968) and Li (2000). The measurement and calculation ofSOD activity were based on the photochemical method described byConstantine and Stanley (1977) with some modifications. The aboveexperiment materials were the roots of six cultivars.

2.3. Statistics

The mean, standard deviation, and statistical differences amongcultivars at different freezing temperatures and periods of time and statis-tical differences among different temperatures and exposure periods inthe same cultivar were all calculated using Duncan's multiple range test(Duncan, 1955) with SPSS 17.0 (SPSS Inc., Chicago, Illinois).

Several studies (Dong et al., 2002; Cui et al., 2009; Zhao et al., 2011)confirmed that REC and plant cold tolerance are negatively correlated,and they considered that the poorer the cold resistance of a plant, thehigher its REC. In this study, correlation analysis among six cold resis-tance indices of different temperatures and different exposure periodswas done firstly. And the indices whose correlation between it andREC is significant at the 0.05 level and 0.01 level were extracted tocalculate the contribution rate (Ci) with principal component analysis.These above processes were also achieved by SPSS 17.0.

The membership function method was used for the comprehensiveevaluation of the six herbaceous peony cultivars using a slightly modi-fiedmethod of Liu et al. (2007) and Lü et al. (2008). At first, the specificmembership function values were calculated with Eq. (1) (if the corre-lation result between the index and RECwas positive) or with Eq. (2) (ifthe correlation result between the index and REC was negative):

Xij ¼ X−Xminð Þ= Xmax−Xminð Þ ð1Þ

Xij ¼ Xmax−Xð Þ= Xmax−Xminð Þ ð2Þ

whereX is the value of character j for cultivar i, Xmin is theminimumvalueof character j among allmeasurements for cultivar i, Xmax is themaximumvalue of character j among all measurements for cultivar i, and Xij is themembership value of cold resistance for cultivar i and character j.

For each cultivar, the integrated index (I)was calculatedwith Eq. (3)(multiplication and addition):

I ¼ Σ Ci � Xij

� �: ð3Þ

The values of ‘I’ indicated the level of cold resistance, which meantthat the lower the value of a cultivar, the more cold resistance it had.

3. Results

Starting from the −6 °C treatment, the REC increased with the low-ering temperature with the best differentiation among cultivars at the−24 °C treatment (Table 2). With a further decline in temperature, thechanges in the REC slowed down and the differences became less. TheREC of ‘Pink Hawaiian Coral’ was significantly different (P ≤ 0.05) fromthat of the other five cultivars from 0 °C to −36 °C (except −6 °C),and it was the first to reach 50% at −24 °C. In addition, the REC of thesix cultivars increased as time passed at low temperature, with a consis-tent significant difference (P≤ 0.05) between ‘Pink Hawaiian Coral’ andthe other five cultivars.

As temperatures decreased, changes in the soluble sugar concentra-tion fluctuated, but generally showed an increasing trend (Table 3).There was no significant difference between ‘Fen Yu Nu’ and ‘PinkHawaiian Coral’ frombeginning to end, and the soluble sugar concentra-tion of these two cultivarswas lower than that of the other cultivars. Thesoluble sugar concentration of ‘Da Fu Gui’, ‘Fen Yu Nu’ and ‘Kansas’remained stable over time at −20 °C, while ‘Monsieur Jules Elie’ and‘Taff’ increased and ‘Pink Hawaiian Coral’ fluctuated.

As can be seen from Table 4, the soluble protein concentration roseunder low temperature stress. However, relative to the increase between−36 °C and 0 °C, the differences were so big that ‘Taff’ had increased by131.83% while ‘Da Fu Gui’ had only increased by 13.85%. From 15 h to35 h at −20 °C, the soluble protein concentration of the same cultivarchanged very little.

The proline concentration of all six cultivars declined from 0 °C to−18 °C in numbers (Table 5). As the temperature decreased furtherto −30 °C and −36 °C, only the proline concentration of ‘Kansas’increased significantly (P ≤ 0.05). Additionally, the proline concentra-tion was significantly (P ≤ 0.05) higher in ‘Kansas’ and ‘Taff’ than thatin the other cultivars at all temperatures.

The MDA concentration most cultivars showed an increasingtrend as temperature declined (Table 6). ‘Monsieur Jules Elie’ was anexception because the value decreased after peaking at −24 °C. Thedifferences between each cultivar becamemore obvious when the tem-perature dropped to −18 °C, and especially at −30 °C, these differ-ences being significant (P ≤ 0.05). At −36 °C, the MDA concentrationof ‘Da Fu Gui’, ‘Fen Yu Nu’ and ‘Pink Hawaiian Coral’ increased morethan 7-fold compared to the respective values at 0 °C. TheMDA concen-tration of ‘Pink Hawaiian Coral’ was significantly different from that ofthe other five cultivars from 5 h to 35 h at−20 °C.

The SOD activity of all cultivars showed an up-and-down trend astemperature decreased (Table 7). By statistical test, we know that theSOD activity of ‘Taff’ peaked at −6 °C while the other five cultivarspeaked at −12 °C. This activity reached a trough at −30 °C except for‘Monsieur Jules Elie’ (the value at−30 °C was higher than that at 0 °C,5.4 U·g−1). At−20 °C, the SOD activity of ‘Taff’ increased continuouslywith the passage of time. In contrast, the SOD activity of ‘Fen Yu Nu’,‘Kansas’ and ‘Pink Hawaiian Coral’ declined at first, reaching lowestvalues at 15 h, and then increased significantly. From 5 h to 25 h, theSODactivity of ‘Fen YuNu’wasminimumandwas significantly differentfrom that of the other five cultivars (P ≤ 0.05).

The measured indices were well correlated with each other. The RECwas positively correlated with soluble sugar, soluble protein and MDAand was negatively correlated with proline and SOD significantly(P ≤ 0.01) at different temperatures (Table 8). While during differentexposure periods, the REC was positively correlated with soluble protein(P ≤ 0.05), MDA and SOD (P ≤ 0.01) (Table 9). The former five indicesand the latter three indices were extracted to calculate the contributionrate, and it showed that soluble sugar, soluble protein, proline, MDAand SOD at different temperatures, while soluble protein, MDA and SODduring different exposure times were contributed from high to low(Table 10).

Based on themembership function values (Table 11), the integratedindices were calculated with Eq. (3), and the cold resistance of the six

Table 2Relativ ectric conductivity (%) of underground buds of six herbaceous peony culti s exposed to different temperatures and different periods of exposure to sub-freezing cond s.

Culti s Temperatures Exposure perio −20 °C

0 °C −6 °C −12 °C 18 °C −24 °C −30 °C −36 °C 5 h 15 h 25 h 35 h

‘Da F ui’ 20.6 ± 2.4 a/ay 21.3 ± 4.5 ab/a 23.5 ± 2.8 a/a 7.2 ± 4.7 a/ab 33.0 ± 2.2 a/b 49.9 ± 5.5 a/c 53.2 ± 1.6 a/c 27.3 ± 2.8 ab/ 30.6 ± 1.3 ab/ab 35.1 ± 4.6 a/bc 39.3 ± 4.0 ab/c‘Fen Nu’ 18.6 ± 3.1 a/a 20.0 ± 2.4 a/a 22.7 ± 1.9 a/ab 7.2 ± 4.3 a/b 34.6 ± 5.3 ab/c 50.8 ± 1.5 ab/d 55.2 ± 4.5 ab/d 25.9 ± 4.0 a/a 28.0 ± 1.8 a/ab 32.5 ± 2.8 a/b 37.7 ± 1.5 a/c‘Mon ur Jules Elie’ 20.2 ± 1.2 a/a 22.3 ± 3.1 ab/a 24.8 ± 3.0 a/ab 9.7 ± 1.8 a/b 39.6 ± 3.0 bc/c 48.7 ± 4.3 a/d 52.1 ± 5.2 a/d 34.4 ± 2.5 b/a 35.9 ± 5.3 bc/a 39.1 ± 1.4 ab/ab 44.9 ± 3.1 c/b‘Kans 19.3 ± 3.0 a/a 21.8 ± 2.2 ab/ab 25.4 ± 1.3 a/b 1.6 ± 4.8 a/c 43.0 ± 2.0 c/d 54.1 ± 1.4 ab/e 54.4 ± 4.8 ab/e 33.8 ± 5.4 b/a 36.7 ± 3.8 bc/ab 38.3 ± 3.8 ab/ab 42.1 ± 0.8 abc/b‘Taff’ 21.0 ± 1.2 a/a 18.4 ± 2.5 a/a 23.4 ± 2.0 a/a 9.1 ± 4.1 a/b 41.7 ± 2.8 c/c 50.0 ± 3.1 a/d 52.6 ± 3.2 a/d 33.6 ± 4.0 b/a 39.9 ± 4.2 c/ab 42.0 ± 2.9 b/b 43.3 ± 3.9 bc/b‘Pink waiian Coral’ 27.9 ± 0.8 b/a 26.6 ± 1.6 b/a 32.9 ± 2.0 b/b 0.5 ± 3.2 b/c 50.5 ± 3.7 d/d 56.9 ± 2.0 b/e 60.7 ± 2.6 b/e 49.3 ± 2.8 c/a 54.8 ± 3.3 d/ab 56.2 ± 4.3 c/b 57.5 ± 2.3 d/b

yDiffere letters in front of ‘/’ indicate significant differences between cultivars at the e temperature according to Duncan'smultiple range test (DMRT) at P ≤ 0.05 (within a colum while different letters behind ‘/’ indicate significant differencesbetwee emperatures within the same cultivar according to DMRT at P ≤ 0.05 (wit a row).zDiffere letters in front of ‘/’ indicate significant differences between cultivars at the e exposure period according to DMRT at P ≤ 0.05 (within a column)while different letters b nd ‘/’ indicate significant differences between exposure periodswithin same cultivar according to DMRT at P ≤ 0.05 (within a row).

Table 3Soluble gar concentration (%) of roots of six herbaceous peony cultivars exposed t ifferent temperatures and different periods of exposure to sub-freezing conditions.

Culti s Temperature Exposure od to −20 °C

0 °C −6 °C −12 °C 18 °C −24 °C −30 °C −36 °C 5 h 15 h 25 h 35 h

‘Da F ui’ 8.8 ± 0.2 b/ay 8.0 ± 0.3 a/a 9.7 ± 0.1 b/b 0.2 ± 0.5 b/bc 10.9 ± 0.9 ab/c 12.1 ± 0.6 bc/d 12.2 ± 0.2 bc/d 11.3 ± 0. az 11.9 ± 1.6 bc/a 12.2 ± 0.6 ab/a 13.2 ± 0.8 c/a‘Fen Nu’ 7.5 ± 0.4 ab/a 7.7 ± 0.8 a/a 8.5 ± 0.4 a/ab 9.2 ± 0.4 ab/bc 10.4 ± 1.1 a/d 9.0 ± 0.3 a/b 10.4 ± 0.9 ab/d 10.9 ± 0. a 11.0 ± 0.1 b/a 12.0 ± 1.2 ab/a 12.1 ± 0.6 b/a‘Mon ur Jules Elie’ 8.1 ± 1.0 ab/a 8.9 ± 0.3 ab/ab 8.8 ± 0.2 ab/ab 0.4 ± 0.4 b/bc 11.5 ± 0.8 abc/cd 12.6 ± 1.3 c/de 13.8 ± 1.7 c/e 11.5 ± 0. /a 11.9 ± 0.2 bc/a 12.9 ± 0.5 b/b 13.9 ± 0.3 c/c‘Kans 8.3 ± 1.1 b/a 9.4 ± 0.1 b/a 9.3 ± 0.1 ab/a 2.1 ± 0.9 c/b 12.8 ± 0.1 c/b 12.9 ± 0.9 c/b 12.5 ± 0.9 bc/b 13.0 ± 0. a 13.0 ± 0.2 c/a 13.4 ± 1.7 b/a 14.1 ± 0.4 c/a‘Taff’ 6.6 ± 1.2 a/a 8.9 ± 0.0 ab/b 11.1 ± 0.7 c/c 1.6 ± 0.4 c/c 12.1 ± 0.3 bc/cd 10.7 ± 1.2 ab/bc 13.9 ± 2.1 c/d 12.6 ± 0. /a 12.9 ± 0.4 c/ab 13.3 ± 0.5 b/ab 13.9 ± 0.3 c/b‘Pink waiian Coral’ 7.9 ± 0.1 ab/a 8.4 ± 1.2 ab/a 9.0 ± 1.2 ab/abc 8.7 ± 0.9 a/ab 10.3 ± 0.3 a/bc 10.6 ± 0.7 ab/c 9.5 ± 1.1 a/abc 9.0 ± 1. a 9.7 ± 0.6 a/ab 10.5 ± 0.6 a/b 10.3 ± 0.6 a/ab

yDiffere letters in front of ‘/’ indicate significant differences between cultivars at the e temperature according to Duncan'smultiple range test (DMRT) at P ≤ 0.05 (within a colu while different letters behind ‘/’ indicate significant differencesbetwee emperatures within the same cultivar according to DMRT at P ≤ 0.05 (wit a row).zDiffere letters in front of ‘/’ indicate significant differences between cultivars at the e exposure period according to DMRT at P ≤ 0.05 (within a column)while different letters b nd ‘/’ indicate significant differences between exposure periodswithin same cultivar according to DMRT at P ≤ 0.05 (within a row).

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Table 4Soluble protein concentration (μg/g) of roots of six herbaceous peony cultivars exposed to different temperatures and different periods of exposure to sub-freezing conditions.

Cultivars Temperature Exposure period to −20 °C

0 °C −6 °C −12 °C −18 °C −24 °C −30 °C −36 °C 5 h 15 h 25 h 35 h

‘Da Fu Gui’ 12.6 ± 0.4 c/cy 9.5 ± 0.9 a/ab 9.1 ± 0.3 a/a 12.9 ± 0.2 a/c 10.5 ± 0.7 a/b 12.5 ± 0.2 b/c 14.4 ± 0.9 b/d 12.3 ± 0.2 a/az 12.2 ± 0.3 a/a 12.4 ± 0.5 a/a 12.4 ± 0.6 a/a‘Fen Yu Nu’ 10.6 ± 0.5 b/a 12.6 ± 0.7 b/b 14.3 ± 1.0 bc/cd 14.8 ± 0.4 b/d 15.3 ± 0.8 b/d 12.9 ± 1.3 b/bc 13.3 ± 0.3 ab/bc 15.3 ± 0.4 c/a 15.3 ± 0.8 b/a 15.9 ± 1.0 b/a 16.1 ± 0.1 b/a‘Monsieur Jules Elie’ 10.8 ± 0.8 b/b 8.7 ± 0.6 a/a 9.3 ± 1.3 a/a 14.7 ± 0.6 b/c 14.7 ± 0.3 b/c 16.9 ± 0.2 c/d 18.1 ± 0.3 c/d 15.2 ± 0.4 bc/a 15.5 ± 1.1 b/a 15.8 ± 0.1 b/a 15.9 ± 0.7 b/a‘Kansas’ 11.5 ± 0.4 bc/b 12.6 ± 0.9 b/bc 13.3 ± 0.8 b/cd 13.0 ± 0.7 a/cd 9.7 ± 0.3 a/a 13.0 ± 0.8 b/cd 14.1 ± 0.7 ab/d 14.2 ± 0.9 b/a 14.9 ± 0.6 b/a 15.2 ± 0.5 b/ab 16.3 ± 0.4 b/b‘Taff’ 9.1 ± 0.6 a/a 13.1 ± 0.7 b/b 15.1 ± 0.2 c/c 13.3 ± 0.8 a/b 14.3 ± 0.5 b/c 16.5 ± 0.3 c/d 21.0 ± 0.3 d/e 17.0 ± 0.6 d/a 18.2 ± 0.7 c/ab 18.4 ± 0.4 c/ab 19.5 ± 1.1 c/b‘Pink Hawaiian Coral’ 10.8 ± 1.1 b/a 13.0 ± 0.3 b/b 17.2 ± 0.6 d/d 17.3 ± 1.0 c/d 15.3 ± 0.3 b/c 10.7 ± 0.9 a/a 13.0 ± 0.9 a/b 15.3 ± 0.6 c/a 15.0 ± 1.6 b/a 15.6 ± 0.9 b/a 15.4 ± 0.4 b/a

yDifferent letters in front of ‘/’ indicate significant differences between cultivars at the same temperature according to Duncan'smultiple range test (DMRT) at P ≤ 0.05 (within a column)while different letters behind ‘/’ indicate significant differencesbetween temperatures within the same cultivar according to DMRT at P ≤ 0.05 (within a row).zDifferent letters in front of ‘/’ indicate significant differences between cultivars at the same exposure period according to DMRT at P ≤ 0.05 (within a column)while different letters behind ‘/’ indicate significant differences between exposure periodswithin the same cultivar according to DMRT at P ≤ 0.05 (within a row).

Table 5Proline concentration (μg/g) of roots of six herbaceous peony cultivars exposed to different temperatures and different periods of exposure to sub-freezing conditions.

Cultivars Temperature Exposure period to −20 °C

0 °C −6 °C −12 °C −18 °C −24 °C −30 °C −36 °C 5 h 15 h 25 h 35 h

‘Da Fu Gui’ 154.0 ± 23.8 bc/cy 99.7 ± 14.2 a/b 61.1 ± 5.7 a/a 58.6 ± 11.4 a/a 69.7 ± 13.0 b/a 78.5 ± 5.8 b/ab 78.5 ± 2.7 b/ab 59.0 ± 10.9 ab/az 60.1 ± 11.0 ab/a 75.9 ± 3.3 b/ab 91.7 ± 7.6 b/b‘Fen Yu Nu’ 112.1 ± 16.2 a/c 100.2 ± 10.3 a/bc 86.7 ± 4.5 b/b 54.6 ± 6.8 a/a 42.4 ± 3.6 a/a 42.7 ± 1.8 a/a 47.7 ± 4.1 a/a 50.3 ± 6.5 a/a 45.8 ± 7.2 a/a 45.8 ± 2.6 a/a 48.6 ± 4.9 a/a‘Monsieur JulesElie’

118.5 ± 8.8 a/d 95.8 ± 3.2 a/c 78.0 ± 5.4 ab/b 77.0 ± 7.2 a/b 63.3 ± 0.6 b/a 69.4 ± 0.7 b/ab 74.3 ± 1.5 b/b 70.7 ± 3.6 b/a 76.3 ± 6.5 b/ab 92.2 ± 9.8 b/bc 108.1 ± 13.1 c/c

‘Kansas’ 221.7 ± 13.2 d/c 199.0 ± 18.0 d/c 169.6 ± 11.6 d/b 116.9 ± 24.0 b/a 98.9 ± 15.7 c/a 106.7 ± 15.3 c/a 154.2 ± 8.1 c/b 122.1 ± 8.1 c/b 116.2 ± 16.0 c/ab 97.8 ± 6.9 b/a 129.5 ± 4.9 d/b‘Taff’ 178.9 ± 12.9 c/d 164.3 ± 9.3 c/cd 161.4 ± 16.2 d/bcd 140.5 ± 8.1 c/ab 146.6 ± 9.5 d/abc 134.6 ± 5.4 d/a 151.1 ± 14.0 c/abc 141.3 ± 13.1 d/a 141.6 ± 3.2 d/a 160.8 ± 24.3 c/ab 176.2 ± 7.4 e/b‘Pink HawaiianCoral’

139.5 ± 22.7 ab/d 135.6 ± 4.9 b/d 111.9 ± 8.8 c/c 77.1 ± 11.3 a/a 72.3 ± 3.2 b/a 104.5 ± 13.8 c/bc 83.9 ± 6.7 b/ab 71.6 ± 5.1 b/a 65.9 ± 8.0 b/a 77.6 ± 7.4 b/ab 89.0 ± 9.9 b/b

yDifferent letters in front of ‘/’ indicate significant differences between cultivars at the same temperature according to Duncan'smultiple range test (DMRT) at P ≤ 0.05 (within a column)while different letters behind ‘/’ indicate significant differencesbetween temperatures within the same cultivar according to DMRT at P ≤ 0.05 (within a row).zDifferent letters in front of ‘/’ indicate significant differences between cultivars at the same exposure period according to DMRT at P ≤ 0.05 (within a column)while different letters behind ‘/’ indicate significant differences between exposure periodswithin the same cultivar according to DMRT at P ≤ 0.05 (within a row).

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able 6DA concentration (μmol/g) of roots of six herbaceous peony cultivars exposed to d rent emperatures and different periods of exposure to sub-freezing conditions.

Cultivars Temperature Exposure per d to −20 °C

0 °C −6 °C −12 °C − °C −24 °C −30 °C −36 °C 5 h 15 h 25 h 35 h

‘Da Fu Gui’ 0.9 ± 0.1 bc/ay 2.5 ± 0.3 c/c 1.9 ± 0.3 cd/b 1.9 0.0 a/b 2.8 ± 0.1 b/c 4.6 ± 0.4 d/d 7.2 ± 0.2 d/e 2.6 ± 0.3 c/a 3.0 ± 0.2 b/b 2.6 ± 0.3 c/ab 2.2 ± 0.3 b/a‘Fen Yu Nu’ 0.4 ± 0.0 a/a 2.0 ± 0.2 b/c 1.3 ± 0.2 b/b 1.8 0.2 a/c 3.4 ± 0.3 c/d 5.3 ± 0.1 e/e 7.1 ± 0.0 d/f 2.1 ± 0.1 b/b 2.2 ± 0.1 a/b 2.1 ± 0.1 ab/b 1.8 ± 0.1 a/a‘Monsieur Jules Elie’ 0.7 ± 0.1 b/a 1.2 ± 0.2 a/b 1.5 ± 0.1 bc/c 1.5 0.2 a/c 2.7 ± 0.1 b/e 1.9 ± 0.1 a/d 1.9 ± 0.1 a/d 1.8 ± 0.1 a/a 1.8 ± 0.3 a/ab 1.9 ± 0.0 a/b 1.5 ± 0.2 a/a‘Kansas’ 0.8 ± 0.2 b/a 1.1 ± 0.2 a/a 2.3 ± 0.3 e/b 2.5 0.3 b/b 3.1 ± 0.1 b/c 3.9 ± 0.3 c/d 4.5 ± 0.3 c/e 2.7 ± 0.1 c/b 2.9 ± 0.1 b/b 2.3 ± 0.2 abc/a 2.4 ± 0.3 b/a‘Taff’ 0.8 ± 0.1 b/a 1.2 ± 0.3 a/b 0.9 ± 0.1 a/a 1.5 0.2 a/b 2.3 ± 0.2 a/c 2.6 ± 0.1 b/c 3.6 ± 0.3 b/d 2.1 ± 0.1 b/a 2.3 ± 0.1 a/b 2.4 ± 0.4 bc/b 1.9 ± 0.1 a/a‘Pink Hawaiian Coral’ 1.1 ± 0.1 c/a 2.9 ± 0.2 d/c 2.0 ± 0.1 de/b 5.7 0.5 c/d 7.7 ± 0.2 d/e 9.6 ± 0.2 f/f 11.4 ± 0.4 e/g 6.4 ± 0.1 d/a 6.7 ± 0.6 c/a 6.7 ± 0.3 d/a 6.8 ± 0.2 c/a

Different letters in front of ‘/’ indicate significant differences between cultivars at the e te perature according toDuncan'smultiple range test (DMRT) at P ≤ 0.05 (within a colum while different letters behind ‘/’ indicate significant differencesetween temperatures within the same cultivar according to DMRT at P ≤ 0.05 (wi a ro ).Different letters in front of ‘/’ indicate significant differences between cultivars at the e ex osure period according to DMRT at P ≤ 0.05 (within a column)while different letters be nd ‘/’ indicate significant differences between exposure periodsithin the same cultivar according to DMRT at P ≤ 0.05 (within a row).

able 7OD activity (U·g−1) of roots of six herbaceous peony cultivars exposed to different per ures and different periods of exposure to sub-freezing conditions.

Cultivars Temperature Exposure perio to −20 °C

0 °C −6 °C −12 °C 18 ° −24 °C −30 °C −36 °C 5 h 15 h 25 h 35 h

‘Da Fu Gui’ 117.0 ± 8.0 a/by 181.8 ± 8.1 de/c 250.9 ± 18.0 c/e 0.6 4.6 c/d 166.8 ± 3.3 b/c 95.5 ± 6.5 a/a 124.0 ± 3.4 bc/b 145.3 ± 5.8 c/a 134.0 ± 5.6 bc/a 173.1 ± 11.0 a/b 238.1 ± 3.9 d/c‘Fen Yu Nu’ 109.3 ± 7.6 a/b 126.9 ± 5.6 b/c 211.0 ± 5.2 b/e 3.7 6.4 b/d 116.7 ± 10.5 a/bc 82.9 ± 6.6 a/a 114.2 ± 10.8 b/bc 101.4 ± 8.1 a/b 84.0 ± 2.2 a/a 160.4 ± 13.9 a/c 209.1 ± 8.8 ab/d‘Monsieur Jules Elie’ 113.7 ± 6.8 a/a 189.7 ± 12.5 e/c 244.2 ± 14.6 c/d 5.6 5.5 c/c 193.3 ± 5.5 c/c 119.1 ± 10.5 b/a 169.5 ± 3.9 d/b 172.8 ± 8.1 d/a 164.0 ± 15.0 d/a 204.4 ± 7.5 b/b 256.4 ± 13.4 e/c‘Kansas’ 141.2 ± 15.3 b/cd 154.2 ± 5.8 c/d 204.3 ± 6.3 b/e 9.6 11.1 a/d 125.6 ± 12.4 a/bc 92.7 ± 3.6 a/a 122.1 ± 11.9 bc/b 147.0 ± 12.0 c 125.0 ± 12.3 b/a 196.3 ± 8.6 b/c 230.1 ± 11.4 cd/d‘Taff’ 157.3 ± 11.8 b/bc 174.0 ± 2.9 d/d 169.9 ± 14.5 a/cd 8.3 6.3 a/b 129.8 ± 4.6 a/a 123.6 ± 6.5 b/a 131.4 ± 3.0 c/a 121.4 ± 4.3 b/a 147.4 ± 10.0 cd/b 203.2 ± 5.3 b/c 218.7 ± 7.1 bc/d‘Pink Hawaiian Coral’ 105.7 ± 6.2 a/bc 108.9 ± 6.5 a/c 180.0 ± 9.1 a/e 3.2 6.0 b/d 153.5 ± 7.1 b/d 84.0 ± 7.2 a/a 95.0 ± 9.6 a/ab 171.1 ± 5.0 d/b 144.0 ± 6.6 c/a 175.1 ± 13.2 a/b 200.3 ± 9.6 a/c

Different letters in front of ‘/’ indicate significant differences between cultivars at the e ex osure period according to DMRT at P ≤ 0.05 (within a column)while different letters be nd ‘/’ indicate significant differences between exposure periodsithin the same cultivar according to DMRT at P ≤ 0.05 (within a row).Different letters in front of ‘/’ indicate significant differences between cultivars at the e te perature according to Duncan'smultiple range test (DMRT) at P ≤ 0.05 (within a colum while different letters behind ‘/’ indicate significant differencesetween temperatures within the same cultivar according to DMRT at P ≤ 0.05 (wit a ro ).

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TM

y

bz

w

TS

y

wz

b

iffe

samthinsam

tem

−

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sam

samhin

t

18

±±±±±±

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p

mw

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bz

b

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hi

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Table 8Correlation analysis among six cold resistance indices of different temperatures.

Cold resistance indices REC Soluble sugar Soluble protein Proline MDA SOD

REC 1Soluble sugar 0.638⁎⁎ 1Soluble protein 0.423⁎⁎ 0.448⁎⁎ 1Proline −0.342⁎⁎ −0.150 −0.072 1MDA 0.764⁎⁎ 0.232⁎⁎ 0.146 −0.360⁎⁎ 1SOD −0.475⁎⁎ −0.150 −0.092 −0.085 −0.420⁎⁎ 1

⁎⁎ Correlation is significant at the 0.01 level (2-tailed).⁎ Correlation is significant at the 0.05 level (2-tailed).

Table 9Correlation analysis among six cold resistance indices of different periods.

Cold resistance indices REC Soluble sugar Soluble protein Proline MDA SOD

REC 1Soluble sugar −0.225 1Soluble protein 0.270⁎ 0.248⁎ 1Proline 0.185 0.585⁎⁎ 0.562⁎⁎ 1MDA 0.720⁎⁎ −0.657⁎⁎ −0.092 −0.175 1SOD 0.463⁎⁎ 0.407⁎⁎ 0.170 0.327⁎⁎ −0.067 1

⁎⁎ Correlation is significant at the 0.01 level (2-tailed).⁎ Correlation is significant at the 0.05 level (2-tailed).

146 Q. Wang et al. / South African Journal of Botany 94 (2014) 140–148

cultivars could be ranked from high to low: ‘Da Fu Gui’, ‘Fen Yu Nu’,‘Kansas’, ‘Monsieur Jules Elie’, ‘Taff’, and ‘Pink Hawaiian Coral’ (Table 12).

4. Discussion

In this study, we established the correlation between REC and otherindices, and as temperatures decreased and exposure periods to sub-freezing conditions went by, the increasing trend of REC existed. How-ever, the REC of each cultivar did not correspond to the final precedenceof cold hardiness respectively. So the comprehensive evaluation of coldresistance among these cultivars is necessary and the relationshipbetween this experiment and field experiment needs further studyand validation.

Table 10The contribution rate of cold resistance indices of different temperatures and different periods

Cold resistance indices Different temperatures

Contribution rate/% Cumulative contributi

Soluble sugar 36.779 36.779Soluble protein 22.687 59.467Proline 21.639 81.106MDA 10.834 91.940SOD 8.060 100.000

Table 11Membership function value of cold resistance indices of six cultivars.

Cultivars Different temperatures

Soluble sugar Soluble protein Proline

‘Da Fu Gui’ 0.633 0.000 0.808‘Fen Yu Nu’ 0.000 0.594 1.000‘Monsieur Jules Elie’ 0.769 0.562 0.848‘Kansas’ 1.000 0.272 0.018‘Taff’ 0.837 1.000 0.000‘Pink Hawaiian Coral’ 0.113 0.752 0.597

This experiment showed that in spite of a complex process, thesoluble sugar and soluble protein concentration of the six cultivarsgenerally presented an increasing trend as temperature declined andalso with the passage of time, which was similar with previous results(Li, 2008; Ren, 2009; Ju, 2011). For example, Ren (2009) found that thesoluble sugar concentration of the roots of P. suffruticosa ‘Shou An Hong’increased by 28.55% from 0 °C to−30 °C while root soluble protein con-centration increased by 0.0687 mg·g−1. In this study, however, the solu-ble sugar concentration of the roots of P. lactiflora ‘Taff’, which rankedfifthin terms of cold hardiness, rose by 60.47% from 0 °C to−30 °C while sol-uble protein concentration increased by 0.0075 mg·g−1.

More proline was accumulated by cultivars with strong resistance(Ju, 2011). Under our experimental conditions, as temperature declinedfrom 0 °C to −24 °C, proline concentration surprisingly decreased.

.

Different periods

on rate/% Contribution rate/% Cumulative contribution rate/%

0.000 0.00040.873 40.8730.000 40.873

31.544 72.41727.583 100.000

Different periods

MDA SOD Soluble protein MDA SOD

0.354 0.264 0.000 0.169 0.5590.339 0.868 0.555 0.056 0.0000.000 0.000 0.550 0.000 1.0000.230 0.703 0.474 0.169 0.5910.047 0.570 1.000 0.086 0.5591.000 1.000 0.503 1.000 0.558

Table 12Integrated index and relative cold resistance precedence of six herbaceous peony cultivars.

Cultivars ‘Da Fu Gui’ ‘Fen Yu Nu’ ‘MonsieurJules Elie’

‘Kansas’ ‘Taff’ ‘PinkHawaiian Coral’

Tem Per Tem Per Tem Per Tem Per Tem Per Tem Per

Soluble sugar 0.233 0.000 0.000 0.000 0.283 0.000 0.368 0.000 0.308 0.000 0.041 0.000Soluble protein 0.000 0.000 0.135 0.227 0.128 0.225 0.062 0.194 0.227 0.409 0.171 0.205Proline 0.175 0.000 0.216 0.000 0.184 0.000 0.004 0.000 0.000 0.000 0.129 0.000MDA 0.038 0.053 0.037 0.018 0.000 0.000 0.025 0.053 0.005 0.027 0.108 0.315SOD 0.021 0.154 0.070 0.000 0.000 0.276 0.057 0.163 0.046 0.154 0.081 0.154Integrated index 0.674 0.703 1.096 0.926 1.176 1.204Precedence 1 2 4 3 5 6

Tem = temperature. Per = periods.

Appendix TableThe average monthly minimum temperature (°C) of Beijing and Harbin.

City Month

1 2 3 4 5 6 7 8 9 10 11 12

Beijing −9 −6 0 8 14 19 22 21 15 8 0 −6Harbin −24 −20 −10 1 8 15 18 16 9 1 −10 −20

Note: Information based on http://www.weather.gov.cn (National Meteorological Centerof CMA, n.d.).

147Q. Wang et al. / South African Journal of Botany 94 (2014) 140–148

Proline concentration is closely related to the osmotic potential of the so-lution in plants (Chu et al., 1973; Lü andWang, 1993). The excess gener-ation of proline can improve a plant's resistance to osmotic stress (Kaviet al., 1995), although osmoregulation does not rely on only one sub-stance to be complete; in addition to proline, it may involve otherorganic compatible materials as well as inorganic ions such as K+

(Wang et al., 1997). Numerous experiments using plant leaves as theexperimental material showed an increase in proline concentrationwhen leaves were exposed to cold, although the mechanism is morecomplex in roots than in other plant parts. Proline is generally consid-ered to be synthesized in roots, but most of its products are transportedto above-ground parts (Armengaud et al., 2004). Herbaceous peonieshave metamorphosed underground shoots, and the decrease in prolineconcentration with the temperature decline from 0 °C to −24 °C islikely to be the result of various factors. Since to date there are fewstudies on the roots of herbaceous peonies, the specific mechanism ofthis phenomenon needs to be further studied.

Ren (2009) considered the cultivars of P. suffruticosa (tree peony)which had strong cold resistance and a low MDA concentration, whichincreased slowly as temperature declined. In this experiment, theMDA concentration of ‘Pink Hawaiian Coral’, which was the least coldresistant cultivar (Table 12), was higher than that of other cultivars atdifferent temperatures and following different periods of exposure(the only exception being ‘Kansas’ at−12 °C), similar to Ren's study.

At−20 °C, the SOD activities of 5 cultivars decreased from 5 h to 15 hthen increased from 15 h to 35 h (only in ‘Taff’ did SOD activity increaseover the entire period). Normally, the SOD activity of all cultivars shouldincrease immediately under stress, but the decrease from 5 h to 15 h isa somewhat surprising result. It is possible to infer that SOD and superox-ide radicals are in a dynamic process in plants under stress, that the rootsof herbaceous peonies are swollen and that the process needs more timeto react. There are many reports about the influence of lipid peroxidationon plant cell membrane permeability, and many researchers agree thatunder cold stress, the intensification of the lipid peroxidation will causean increase in membrane permeability. The herbaceous peony cultivars'MDA, which acts as an end product of lipid peroxidation, was very likelyto increase when the temperature dropped from 0 °C to−36 °C, a phe-nomenon that has been confirmed in many plants (Jouve et al., 1993;Zhou, 2001; Hara et al., 2003).

Thirteen intersectional hybrids of herbaceous peony × tree peonywere successfully introduced to Beijing from the U.S. by Sun and Cheng(2007). Previous studies (Yao, 2009; Yu et al., 2011b) found that the intro-duced herbaceous peony cut-flower cultivars (including ‘Monsieur JulesElie’, ‘Kansas’, ‘Taff’, and ‘Pink Hawaiian Coral’) were adapted to andbloom normally in Beijing without any special maintenance. But in thisexperiment, ‘Pink Hawaiian Coral’, one of the Hybrid GP cultivars, is theleast cold resistant among the test cultivars. Cold hardiness is related toplant genetic, environmental effects, plant age, and many other factors.Though ‘Pink Hawaiian Coral’ is an interspecific hybrid, its cold resistanceis far inferior to that of the cultivars of Lactiflora GP.

Considering thepopularity of peonies in northern China, especially inHarbin, the highest latitude provincial capital city in China, the average

monthly minimum temperatures of Beijing and Harbin (AppendixTable) and previous studies served as references for the experimentaltemperature treatment. Since the average monthly minimum tempera-ture of Harbin is −24 °C in January, and both Li (2008) and Li (2009)used temperatures that ranged from 0 °C to −40 °C at intervals of−5 °C in their cold-resistance studies of P. rockii in Harbin, the temper-atures used in this study were based on these previous assessments,also taking our experimental conditions into account.

In addition to temperature, plant age, and nutrient supply, the type ofsubstrate and other factors can be controlled in the open field at the timeof planting. The membership function method is commonly used for thecomprehensive evaluation of cold hardiness of plants, including Paeonia(Ju, 2011) and Lagerstroemia (Tang et al., 2012). Such conditions need tobe optimized in order to extend the use of herbaceous peony cultivarsto latitudes higher than those of Beijing, particularly for ‘Da Fu Gui’, ‘FenYu Nu’, ‘Kansas’, ‘Monsieur Jules Elie’, ‘Taff’, and ‘Pink Hawaiian Coral’.

Acknowledgments

This work was financially supported by the Fundamental ResearchFunds for the Central Universities (YX2014-20) and IntroducingAdvanced Technology Project from the State Forestry Administration(2012-4-59).

Appendix A

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