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Article

Effect of Cutting Phenological Stage, Chemical Treatments, and Substrate on Rooting Softwood Cuttings of Tree Peony

by
Dongli Li
1,
Fangyun Cheng
1,*,
Xiwen Tao
2 and
Yuan Zhong
1
1
State Key Laboratory of Efficient Production of Forest Resources, Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, National Engineering Research Center for Floriculture, Peony International Institute, School of Landscape Architecture, Beijing Forestry University, Beijing 100083, China
2
Beijing Guose Peony Technology Co., Ltd., Beijing 102100, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(5), 552; https://doi.org/10.3390/horticulturae11050552
Submission received: 16 April 2025 / Revised: 12 May 2025 / Accepted: 19 May 2025 / Published: 20 May 2025
(This article belongs to the Special Issue Propagation and Flowering of Ornamental Plants)
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Abstract

:
Tree peony is well-known for its ornamental value, medicine function, oil, and edible use. However, the difficulty in propagating tree peony impedes its cultivation and industrial advancement. Softwood cutting is an effective method to promote the propagation of tree peony. This research investigated the effects of several factors (cultivar, cutting phenological stage, auxin type, polyamine, and substrate) on the rooting of softwood cutting in tree peony. The results showed that rooting ability varied with cultivars and cutting phenological stages, with the highest rooting rates being for ‘High noon’ and ‘Jinghua Qingxue’ during the vigorous growth stage, reaching 50% and 53.33%, respectively. IBA 2000 mg·L−1 was optimal for rooting in ‘High noon’ cuttings, with the maximum root number (5.67) and root length (6.3 cm). Putrescine of 1.0 mM could significantly improve the rooting rate and rooting quality of ‘Jinghua Qingxue’ cuttings, which had the highest rooting rate of 54.17% in the cocopeat/perlite substrate (v:v 1:1). Anatomical observation showed that most adventitious roots were generated from callus meristem nodules differentiated from cortical parenchyma cells while a few came from stem bark, as well as integrated root induction. This study is an innovation in and supplement to tree peony propagation research, and a propagation protocol was primarily established for softwood cuttings in tree peony.

1. Introduction

Tree peony (Paeonia sect. Moutan) is a traditional ornamental flower which is also used in medicine, as an oil, and in cosmetology and food science [1,2]. However, the difficulty in propagating tree peony impedes its cultivation and industrial advancement [3]. Softwood cuttings have been widely used in the industrial production of commercial seedlings for many ornamental and crop plants, due to their advantages of a high reproduction coefficient, short growth cycle, and simple operation [4]. Meanwhile, softwood cutting is a potent supplementary method to promote difficult-to-root species propagation and early flowering and fruiting in many plants [5,6,7].
The success of softwood cuttings depends on the formation of adventitious roots (ARs), which is influenced by both endogenous and exogenous factors [8]. Many factors, such as genotypes, phenological stage, plant growth regulators (PGRs), polyamines, and substrates, have been confirmed to be involved in the rooting of cuttings in various plants [9,10,11,12]. The rooting ability of cuttings is closely related to genotypes, phenological stage, and climatic conditions which lead to seasonal variation in woody plants [13,14]. Johnson et al. [10] investigated 20 wild and cultivated Prunus genotypes and found that the plum species rooted at a higher percentage and produced more roots than the peach species, while the almond species generally failed to root. The rooting percentage varied from 0% to 62.5% in diverse cultivars of Pistacia lentiscus L. [15] and exhibited significant differences in seven pomegranate cultivars [7]. As reported, cutting specifications also have an important influence on the rooting of cuttings, including the length, diameter, and the amounts of leaves [5,16,17,18]. Furthermore, many studies have shown that the phenological stages of the stock plants play an important role in the propagation of softwood cuttings, indicating that the homeostasis between root-promoting substances and root-inhibiting substances in the stock plants changes with seasonal variation [14,16]. It was reported that four difficult-to-root rose cultivars (Duchesse d’Angoulême, Hurdals, Maiden’s Blush, and Mousseuse Rouge) showed different rooting abilities at four phenological stages and that all cultivars had the highest rooting percentage at the closed flower buds stage, while none of the cuttings could root during the 10~14 days after petal shedding [5].
Among these exogenous rooting factors, indole-3-butyric acid (IBA) is widely used as an efficient stimulant to promote the rooting of cuttings in many woody plants, including Rosa damascena [19], Sterculia foetida [20], Prunus cerasifera [10], Acer rubrum [21], Photinia x fraseri [22], and Juglans regia [23]. The effect of exogenous IBA application on rooting depends on the rooting capacity of various genotypes [21,24]. Moreover, the role of IBA is supposed to convert into indole-3-acetic acid (IAA) via β-oxidation in peroxisome [25], by which IBA can upregulate the auxin signaling pathway and enhance the accumulation of IAA at the rooting sites to promote rooting [23]. In some cases, IBA alone could not promote the formation of AR in cuttings, while the combination of IBA and NAA resulted in better rooting rate [26,27].
Polyamines (PAs) alone or combined with IBA are beneficial for rooting in many plants, including Corylus avellana [28], Olea europaea [29], Prunus persica x Prunus amygdalus [30], and Rosa damascena [12]. As reported, putrescine (Put), spermidine (Spd) and spermine (Spm) had positive impacts on AR formation in Rosa damascena (difficult-to-root) and Put led to a better rooting performance when used in conjunction with IBA [12]. PAs have been proposed as rooting markers since their endogenous accumulation has been positively correlated with the rooting induction and initiation phases [30,31].
In addition, the type of substrates have a significant effect on rooting parameters in stem cuttings and might be genotype-dependent [32,33,34]. The highest rooting rate of Pinus massoniana cuttings occurred in sand (75.17%), followed by perlite (60.52%), while the rooting parameters, including root number, root length, root diameter and rooting effect index, were superior in perlite than other substrates [11]. Recently, some soilless substrates have been widely used for commercial cuttings, including rockwool, horticultural foam, and aeroponics [33,35,36], among which aeroponics was more effective than other methods due to there being less algae or mold over time [34].
However, few studies have been reported on softwood cutting in tree peony. The test of leaf-bud cuttings was carried out in four cultivars of tree peony and two of them successfully rooted from the base callus two months after the cutting [37]. The rooting rate of five tree peony cultivars varied from 20% to 91% through etiolated softwood cuttings, with the superior substrate of vermiculite [38]. It was found that large amounts of callus tissues were induced at the incision base before rooting and that ARs finally protruded out of callus surface [39,40]. It is important to determine the key factors influencing the rooting efficiency of tree peony softwood cutting. Accordingly, the aim of the study was to evaluate the effects of phenological stage, auxin types, polyamines, and substrates on the rooting of nine tree peony cultivars to improve nursery propagation, and the optimal cutting propagation protocol was summarized.

2. Materials and Methods

2.1. Plant Materials

In 2021–2022, nine cultivars of tree peony (Itoh peony ‘Bartzella’, Paeonia × lemoinei ‘High Noon’, Paeonia rockii ‘Jinghua Qingxue’, ‘Jing Yunguan’, ‘Jinghong’, ‘Jing Zuimei’, ‘Jing Helan’, ‘Gaoyuan Shenghuo’, and Paeonia suffruticosa ‘Luoyang Hong’) were chosen from the plants growing in the nursery of Beijing Guose Peony Garden (40°45′ N, 115°97′ E), Beijing, China. The softwood and semi-lignified cuttings were collected from the current shoots after pruning rejuvenation last fall, then trimmed to 6~8 cm long with 1~2 buds and sterilized with carbendazim for further experiments.

2.2. Cutting Treatments and Experimental Conditions

2.2.1. Cutting Phenological Stage

The cuttings of nine cultivars (refer to Section 2.1) were prepared at different phenological stages from 10 days before flowering (DBF) to 90 days after flowering (DAF). The cuttings were inserted in peat/perlite substrate (v:v 6:4) after the basal parts (2 cm) were soaked in IBA solution (2000 mg·L−1) for 15 min. For each collection time and cultivar, 10 cuttings with 3 replicates were considered.

2.2.2. Plant Growth Regulators

The cuttings were taken from the tree peony ‘High Noon’ at 10 DBF in 2021. The treatment of plant growth regulators was detailed in Table 1. IBA solutions were prepared by dissolving IBA powder (Biorigin, Beijing, China) into distilled water using 1 mol·L−1 of sodium hydroxide. Genpan (active ingredient 2.5% IBA + 2.5% NAA, Sichuan Guoguang Agrochemical Co., Ltd., Chengdu, China) solutions were diluted with distilled water to the corresponding ratio, and rooting powder (active ingredient 0.25%IBA, Rhizopon, Hazerswoude, the Netherlands) was directly used, with water as a control. The basal parts (2 cm) of cuttings were dipped into these solutions for 15 min or into rooting powder for 3 s and they were inserted into the peat/perlite substrate (v:v 6:4). For each treatment, 10 cuttings with 3 replicates were considered.

2.2.3. Polyamines

The experiment into how polyamines influence rooting was conducted in the tree peony ‘Jinghua Qingxue’ at 19 DAF in 2022. In this experiment (Table 2), the basal parts (2 cm) of the cuttings were initially dipped in solutions of putrescine, spermidine, and spermine at three concentrations (0.05 mM, 0.1 mM, and 1.0 mM) for 5 mins and then dipped in IBA solution (2000 mg·L−1) for 15 min with water as a control. Finally, the cuttings were inserted into a perlite/vermiculite substrate (v:v 7:3). For each treatment, 10 cuttings and 3 replicates were considered.

2.2.4. Substrates

The experiment of substrates influencing rooting was conducted on the tree peony ‘Jinghua Qingxue’ at 19 DAF in 2022. In this experiment, the cuttings were treated with IBA 2000 mg·L−1 for 15 min and inserted in seven rooting substrates: (T1) cocopeat, (T2) cocopeat-perlite (v:v 1:1), (T3) cocopeat-perlite (v:v 1:2), (T4) perlite-vermiculite (v:v 7:3), (T5) perlite-peat (v:v 3:1), (T6) perlite-peat (v:v 4:6), (T7) perlite-peat-vermiculite (v:v:v 3:1:1). For each treatment, 10 cuttings with three replications were considered.

2.2.5. Experimental Conditions

All these experiments were conducted in the greenhouse of the Beijing International Flower Port (40°10′ N, 116°47′ E), Beijing, China. An intelligent controlled fog system maintained the optimal climatic conditions (air temperature 18 °C~30 °C and relative humidity 75~90%). The cuttings were artificially water-sprayed every 2 h to keep the leaves moist during the day and covered with a shading net (shading percentage of 70%) that reduced the light intensity. The cuttings were protected by applying fungicides and pesticide every 7 days during rooting: the dilute solutions (800~1000) consisted of carbendazim (Sichuan Guoguang Agrochemical Co., Ltd., Chengdu, China), dimethomorph (Acrobat-BASF, Shanghai, China), mancozeb (Limin Chemical Co., Ltd., Xinyi, China), and imidacloprid (Sichuan Guoguang Agrochemical Co., Ltd., Chengdu, China).

2.3. Evaluation of Rooting Cuttings

Three cuttings were randomly selected for observation every 7 days in each treatment and returned to the substrate for continuous growth. The adventitious roots appeared at the surface of callus or protruded from bark of the stem at approximately 60 days after cutting (DAC). Then, 90 DAC, the rooting parameters were calculated to evaluate the effects of developmental stage, cultivar, plant growth regulators, polyamines, and substrates on the rooting of cuttings, including rooting rate, callus rate, rot rate, average root number, and root length. The calculation formulas were as follows:
Rooting rate = (Number of rooted cuttings/Total number of cuttings) × 100%
Callus rate = (Number of callusing cuttings/Total number of cuttings) × 100%
Rot rate = (Number of rot cuttings/Total number of cuttings) × 100%
Average root number = (Total root number/Total number of rooted cuttings) × 100%
Average root length = (Total root length/Total root number) × 100%

2.4. Morphological and Anatomical Observation on Shoots Development and Rooting Process

To study the developmental stage of shoots and the rooting process of cuttings, the tree peony ‘High Noon’ was chosen for morphological and anatomical observation. Five shoots were collected and recorded at each of the four developmental stages of 10 DBF, 28 DAF, 55 DAF (2022), and 80 DAF in 2021, respectively. Then, the basal part (2 cm) of the cuttings was cut into pieces (2 mm thickness) and put into FAA fixative (90 mL 70% alcohol, 5 mL 38% formaldehyde, and 5 mL acetic acid), and the anatomical structure was studied through paraffin sectioning [41]. During rooting process, the rooting sites of ‘HN’ cuttings at about 60 DAC were cleaned carefully and cut into pieces (2 mm thickness) for paraffin sections to identify the origin of adventitious roots [42]. After staining with safranin O-fast green (Biorigin, Beijing, China), the paraffin sections were sealed with neutral gum and photographed under a SOPTOP optical microscope (Leica, Wetzlar, Germany).

2.5. Statistical Analysis

The data analysis was performed using Excel (2019) (Microsoft Corporation, Redmond, WA, USA) and data were expressed as the mean ± standard deviation. The comparisons between different data were analyzed by one-way analysis of variance (ANOVA) (p < 0.05) and Duncan’s test using SPSS software (version 27.0; IBM, Chicago, IL, USA). The statistical significance level was set at p < 0.05.

3. Results

3.1. Effect of Cutting Phenological Stage on the Rooting of Tree Peony

In the 2021 test, the cuttings of four cultivars could root after we prepared them from shoots at the developmental stages of both 10 DBF and 30 DAF, and only a few ‘HN’ cuttings rooted at 80 DAF (Table 3). The highest rooting rate of ‘HN’ cuttings was obtained from the 28 DAF plants, reaching 50%, followed by rates of 38.33% and 6.67% obtained from the 10 DBF and 80 DAF plants, respectively. Higher rooting rates for the other three cultivars were obtained from 10 DBF plants, achieving rooting rates of 31.67%, 21.67%, and 16.67% for ‘JHQX’, ‘JYG’, and ‘GYSH’, respectively. It was found that the rooting ability of cuttings was consistent with the callus rate and opposite to the rot rate in the four cultivars (Table 3).
In the 2022 test, the rooting rates varied significantly, ranging from 0 to 53.33% at different developmental stages among the nine cultivars (Table 3). Obviously, higher rooting rates of these cuttings were noticed at around 20~40 DAF, but no rooting was newly generated beyond 60 DAF. Among these cultivars, the highest rooting rate was obtained from ‘JHQX’ at 19 DAF, achieving 53.33%, followed by a rate of 30% obtained from ‘BZL’ at 36 DAF (Table 3). Along with the maturity of shoots for softwood cuttings, the rates of rooting and callusing in these cultivars gradually decreased while the rot rates increased (Table 3), indicating that the pivotal role of developmental stages of shoots in the rooting of tree peony softwood cuttings.

3.2. Effect of Plant Growth Regulators on Rooting

The rooting rates of ‘HN’ cuttings with all PGR treatments were higher than the control group, as well as the callus rates (Figure 1A). The highest rooting rate was obtained with the use of IBA 2000 mg·L−1 (38.33%), followed by 1000 mg·L−1 (36.67%). A lower rooting rate was noticed for RP (10%), just higher than the control (5.89%). Furthermore, a callus rate of 100% was achieved with IBA 2000 mg·L−1, GP-250, and GP-350. However, the rot rate varied significantly among all the PGR treatments, with the lowest and highest rot rates obtained with IBA 2000 mg·L−1 (6.67%) and GP-150 (60.83%), respectively (Figure 1A). Additionally, PGRs also significantly affected the root number and root length of cuttings. The largest root number occurred in the treatment with RP (8.0), followed by IBA 2000 mg·L−1 (5.67) (Figure 1B). The longest root length was obtained with the treatment of GP-150 (6.6 cm), followed by RP (6.47 cm) and IBA 2000 mg·L−1 (6.37 cm) (Figure 1C). Considering all of these results, IBA 2000 mg·L−1 was the optimal treatment to promote rooting for softwood cuttings in the tree peony ‘High noon’.

3.3. Effect of Polyamines on Rooting

Combined with 2000 mg·L−1 IBA, the rates of rooting and callusing were higher with Put treatments in ‘JHQX’ cuttings, and only few cuttings could root in Spd-0.1 and Spm-1.0 treatments, while the cuttings hardly rooted in the remaining treatments (Figure 2A). The highest rooting rate was obtained by Put-1.0 (25%), followed by Put-0.1 (20.83%) and Put-0.05 (16.67%). Meanwhile a higher callus rate of 95.83% was achieved with Put-1.0 and Put-0.05 compared with other treatments. Additionally, lower rot rates ranged from 41.67% to 45.83% in Put, and higher rot rates reached 70.83~91.67% in the treatments of Spd and Spm as well as in the control group. Furthermore, Put-1.0 could produce the maximum root number and root length, reaching 6.33 and 4.0 cm, respectively (Figure 2B,C), which was more conducive to later growth for cuttings. Therefore, Put-1.0 was suitable for promoting AR formation in ‘JHQX’ softwood cuttings.

3.4. Effect of Substrates on Rooting

The rooting rate and quality varied significantly among the seven substrates in ‘JHQX’ softwood cuttings (Figure 3A–C). Rooting rates obtained from T2, T6, and T4, reaching 54.17%, 53.33%, and 37.5%, respectively, were higher than the rates of 16.67%, 8.33%, and 4.17% obtained from T5, T7, and T1, respectively (Figure 3A), while none of the cuttings could root in substrate T3. Similarly, higher callus rates were obtained from T2, T4, T5, and T6, ranging from 91.67% to 96.67%. Lower callus rates were obtained from other three substrates, ranging from 45.83%~62.5%. Our analysis indicated that the lowest rot rate was obtained from T6 (6.67%), followed by T4 (25%) and T2 (37.5%) (Figure 3A). Furthermore, the maximum root number (8.33) and root length (10.93 cm) were significant in T2, followed by T6 with a root number of 5.33 and a root length of 7.83 cm (Figure 3B,C), indicating that the rooting performance was better in T2 than T6. On the basis of these results, T2 substrate was suitable for callusing and rooting in softwood cuttings of the tree peony ‘JHQX’.

3.5. Softwood Development and Rooting of Softwood Cuttings in Tree Peony

3.5.1. Morphological and Anatomical Observation of ‘HN’ Softwood Development

Based on the results in Section 3.1, the rooting ability of tree peony cuttings was closely associated with the developmental stages of shoots; thus, the current shoots for ‘HN’ softwood cuttings were observed in terms of their morphological and anatomical aspects at different developmental stages in 2021 (Figure 4). We found that the shoot stem was initially very tender and red–purple at 10 DBF (Figure 4A), then turned hard and green at 28 DAF (Figure 1B) and gradually became yellow beyond 55 DAF (Figure 4C,D). Synchronously, the leaves of shoots were primarily not yet unfolded and red–purple at 10 DBF (Figure 4A), gradually unfolded with extension and turned green at 28 DAF, then turned yellow beyond 55 DAF (Figure 4B–D), which indicated that the contents of chlorophyll and carotenoid and photosynthetic capacity of shoots varied with developmental stages [43]. Via further observation through an optical microscope, the primary structure of cuttings stem consisted of the epidermis, cortex, phloem, xylem, and pith from the outside to inside at 10 DBF (Figure 4a), indicating that the differentiation of shoots had not yet been completed and that the cells possessed a high potential differentiation capability. The parts of phloem and xylem were found to be obviously differentiated and thickened at 28 DAF (Figure 4b), when the shoots were at the vigorous growth stage. Beyond 55 DAF, a ring of sclerenchyma [44] was noticed outside the phloem and the xylem layer gradually thickened (Figure 4c,d), which indicated that the shoots were gradually undergoing lignification and that the rooting ability might decrease [24]. As mentioned above, the highest rooting rate of ‘HN’ cuttings was obtained from 28 DAF (50%) and subsequently from 10 DBF (38.33%). Therefore, the developmental stages of shoots played an important role in the adventitious rooting of softwood cutting for tree peony, and the cuttings were more likely to root when prepared from shoots in vigorous growth.

3.5.2. Rooting Process and Rooting Type of ‘HN’ Softwood Cuttings

The rooting process of ‘HN’ cuttings were observed by taking the cuttings prepared at 10 DBF as an example (Figure 5). Initially, the leaflets of ‘HN’ cuttings were purplish red and curled on 0~10 days after cutting (DAC) (Figure 5A), then turned green on 20~30 DAC (Figure 5B) and unfolded after 40 DAC (Figure 5C). Synchronously, the stem of cuttings gradually turned from purplish red to green and yellow (Figure 5A–D). Generally, the callus tissues were primarily formed at the base incision of cuttings on 10 DAC (Figure 5E) and gradually enlarged at 10~50 DAC (Figure 5F); then, the callus nodules were prominently formed and observed on the surface of callus at 56 DAC (Figure 5G). Finally, the adventitious roots protruded from callus nodules of the cutting base or the stem bark of cutting beyond 60 DAC (Figure 5H,I).
We observed that the rooting of the ‘HN’ cuttings was divided into three types. Most adventitious roots generated from callus tissues and a few were generated from stem bark, while some demonstrated integrated root induction (Figure 6A–C). Through section observation on the anatomical structure, no latent root primordia were found in the stem of softwood cuttings (Figure 6D). In the callus-induced rooting type, the adventitious roots were formed from callus meristem nodules which were differentiated from cortical parenchyma cells (Figure 6E). In the bark-derived rooting type, the cells of vascular cambium layer or phloem parenchyma cells dedifferentiated to root primordia and developed into ARs (Figure 6F).

4. Discussion

Rooting of cuttings is a prerequisite for the establishment of post-field transfer survival for plants in the soil [45]. Previous reports showed that tree peony cuttings find it difficult to root and that the rooting capacity of this species is significantly different among cultivars [38,46], although ‘High noon’ softwood cuttings find it relatively easier to root [39]. In this study, nine cultivars demonstrated significant differences in rooting ability, among which ‘High noon’ and ‘Jinghua Qingxue’ demonstrated better rooting parameters than other cultivars. The rooting ability of cuttings varies greatly according to the developmental stages and anatomical structure of plants [7,43]. For this reason, the shoots with higher rooting ability should be collected for cuttings which are more conducive to rooting and survival [43]. We found that the highest rooting rates of ‘High noon’ and ‘Jinghua Qingxue’ cuttings were obtained frox 28 DAF (50%) and 19 DAF (53.33%) in June when the shoots were in a vigorous growth period (Table 3), which was similar to the rooting result of Brunfelsia pauciflora and Cercis siliquastrum [24,47]. It was observed that the phloem and xylem parts of shoots were obviously thickened and that a ring of sclerenchym formed outside the phloem of the ‘HN’ softwood after 55 DAF (Figure 4c,d). It was proposed that when the shoots gradually undergo lignification, root-promoting substances might progressively diminish, while those with inhibitory effects continuously accumulate, which would result in decreased rooting ability [16,23].
Currently, the application of IBA is found to be more effective for promoting AR formation in difficult-to-root cuttings than other plant growth regulators [7,20]. However, the effect of IBA on rooting depends on the genotypes and phenological stages of shoots [5,12,19]. The effective treatments with IBA were 300 mg·L−1 for 1 h in Acer Rubrum [21] and 4000 mg·L−1 for 10 s in Brunfelsia pauciflora [24] for stem cuttings. In this study, the highest rooting rate of ‘High noon’ was obtained using IBA 2000 mg·L−1, followed by 1000 mg·L−1 (Figure 1A). The rooting powder induced better performance in terms of root number and root length, followed by IBA 2000 mg·L−1 (Figure 1B,C). Therefore, the combination of IBA might need to be optimized to improve rooting for softwood cuttings in tree peony.
Polyamines combined with IBA could affect the rooting performance in stem cuttings, which produced higher rooting rate and better root quality than polyamines alone in woody plants [28,48,49]. Wen et al. [48] found that Put was more effective at promoting in the vitro rooting of three tree peony cultivars than Spm and Spd in combination with IBA, while the optimal treatment of Put varied among the three cultivars. The synergistic effects of IBA with Put, Spd, and Spm could all promote the callus rate of cuttings, but the latter two had no significant effects on the rooting of ‘Jinghua Qingxue’ (Figure 2A). Moreover, Put 1.0 mM could induce the maximum rooting rate of 25% along with IBA 2000 mg·L−1, and both the root number and root length reached their best values of 6.33 and 4.0 cm, respectively (Figure 2B,C). The role of PAs in the initiation and induction phases of rooting has been previously reported [49,50], while the interaction of PAs and auxin has been hypothesized during the AR formation of stem cuttings [13,28]. Moreover, a lower content of free PAs was observed in difficult-to-root species than in easy-to-root species, indicating the positive role of PAs in rooting [13,29]. In the future, the combination of PAs and IBA treatments should be adjusted to improve rooting rates and reduce rot rates in tree peony softwood cuttings, and the effect of PAs on rooting also need to be analyzed further.
The optimal substrate should have high water holding capacity and sufficient porosity for achieving the right air/water balance [14], while the ideal substrates depend on plant genotypes, the cutting’s phenological stage, and the auxin types [32,51,52]. Recently, propagators have tended to use a combination of organic (cocopeat, peat, peat moss, sand) and mineral components (perlite, vermiculite, rockwool) as substrates for soilless cultivation [11,51,52,53,54,55]. Abass et al. [51] found that the rooting rates and root number both performed better in sand/perlite (v:v 1:1) and sand/vermiculite (v:v 1:1) combined with IBA 3000 mg·L−1 for cuttings of Pedilanthus tithymaloids. Ghimire et al. [52] reported that a substrate of perlite/vermiculite (v:v 1:1) with IBA 2000 mg·L−1 was more effective than soil in promoting rooting in cuttings of Chrysanthemum indicum L. In this study, IBA 2000 mg·L−1 combined with T2, T6, and T4 could significantly increase the rooting rates of ‘Jinghua Qingxue’ cuttings, reaching 54.17%, 53.33%, and 37.5%, respectively (Figure 3A). Similarly, higher callus rates were obtained from T2, T4, T5, and T6, ranging from 91.67% to 96.67% (Figure 3A). As reported, perlite has higher water holding capacity and pores and lower bulk density than vermiculite [56], so substrates including perlite were more conducive to rooting [57], which was consistent with our results. It was revealed that the rot rate of T2 was higher than T6, but root number and length performed better in T2 than in T6 (Figure 3B,C). Better root formation was attributed to higher water holding capacity and better aeration for cutting substrates [52]. Based on the analysis, the T2 substrate was more suitable for promoting rooting of softwood cutting in the tree peony ‘JHQX’. In the future, the physical and chemical properties of substrates should be analyzed before the cutting is planted; meanwhile more combinations of different substrate mixtures and auxin application need to be tested to increase the rooting performance of tree peony softwood cuttings.
AR formation can be divided into three types, namely callus induction, bark-derived rooting, and integrated rooting induction [58,59]. In this study, most cuttings rooted from the callus, but a few were derived from bark, and some were of the integrated rooting type, which was consistent with the results of Chen et al. [39]. As reported by Cheng et al. [60], ARs mainly originated from meristematic nodules of the callus which were formed from the differentiation of cortical parenchyma cells. We found that the callus tissues of cuttings were primarily formed on 10 DAC and gradually enlarged at 10~50 DAC during rooting; ARs then protruded through the callus surface beyond at least 60 DAC (Figure 5). Through the anatomical observation (Figure 6), no root primordia were found in the stem of the tree peony before cutting, and root primordia originated from callus meristem nodules, in accordance with the result of Cheng et al. [60] Additionally, roots were also derived from vascular cambium cells or phloem parenchyma cells, which was similar to the research of Meng et al. [40] and Wen et al. [42]. These findings are consistent with the results of previous studies [37,38,39,40], indicating that the rooting of tree peony cuttings mainly belongs to the callus induction type and that tree peony is a difficult-to-root species.
According to the present study, an attempt at acclimation and transplanting for seedlings was made using the rooted cuttings, and a propagation protocol was established for softwood cuttings of tree peony, as illustrated in Figure 7. Firstly, the current shoots after pruning rejuvenation were collected and pruned for cuttings, then sterilized with fungicides (e.g., carbendazim) and inserted into the appropriate substrate (Figure 7A–C). The cuttings were artificially water-sprayed and we applied fungicides and pesticide during the rooting, as described in Section 2.2 (Figure 7D). At about 100 DAC, the cuttings could form root balls and were transplanted into suitable pots (Figure 7E,F). During the rooting cuttings’ growth, a water supply and fertilizer input were required regularly. After one year of being transplanted, vigorous roots and newly grown leaves could be seen (Figure 7G). After 30 months of being transplanted, flower buds appeared at the shoot apex (Figure 7H). Consequently, softwood cutting could be used as an effective method to promote AR formation and improve reproductive efficiency for tree peony propagation.

5. Conclusions

The study showed that two cultivars of tree peony, namely ‘High noon’ and ‘Jinghua Qingxue’, demonstrated better rooting ability than other cultivars. The highest rooting rates of both cultivars were obtained from shoots at the vigorous developmental stages in June. Moreover, IBA 2000 mg·L−1 was proved to significantly promote the rooting of ‘High noon’ cuttings. Put 1.0 mM combined with IBA 2000 mg·L−1 could induce a higher rooting rate and a better rooting index for ‘Jinghua Qingxue’ cuttings, alongside a recommended substrate of cocopeat/perlite (v:v 1:1). The cuttings of the tested cultivars mainly demonstrated callus induction rooting, and most ARs were generated from callus meristem nodules differentiated from cortical parenchyma cells. Based on these results, a protocol propagation was primarily established for softwood cuttings of tree peony.

Author Contributions

Conceptualization, D.L. and F.C.; Methodology, D.L., F.C. and Y.Z.; Formal analysis, D.L.; Funding acquisition, F.C.; Investigation, D.L. and X.T.; Resources, X.T. and F.C.; Data curation, D.L., F.C. and Y.Z.; Writing—original draft preparation, D.L. and F.C.; Writing—review and editing, D.L. and F.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key Research and Development Project of China (Grant No. 2020YFD1000503) and the 5·5 Engineering Research & Innovation Team Project of Beijing Forestry University (Grant No. BLRC2023A06).

Data Availability Statement

Data are contained within this article.

Conflicts of Interest

Author Xiwen Tao was employed by the Beijing Guose Peony Technology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Effect of plant growth regulators on rooting parameters of ‘High Noon’ cuttings. (A–C) represent the rates of rooting/callus/rot, root number, and root length with different plant growth regulators on the rooting of ‘High Noon’ cuttings, respectively. Different letters indicate significant differences at the p level below 0.05.
Figure 1. Effect of plant growth regulators on rooting parameters of ‘High Noon’ cuttings. (A–C) represent the rates of rooting/callus/rot, root number, and root length with different plant growth regulators on the rooting of ‘High Noon’ cuttings, respectively. Different letters indicate significant differences at the p level below 0.05.
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Figure 2. Effect of polyamines on rooting parameters of ‘Jinghua Qingxue’ cuttings. (A–C) represent rates of rooting/callus/rot, root number, and root length of different polyamines on the rooting of ‘Jinghua Qingxue’ cuttings, respectively. The cuttings were initially dipped in the solutions of Put, Spd, and Spm at three concentrations (0.05 mM, 0.1 mM, and 1.0 mM) for five mins and then dipped in IBA solution (2000 mg·L−1) for 15 min with water as a control. Different letters indicate significant differences at the p level below 0.05.
Figure 2. Effect of polyamines on rooting parameters of ‘Jinghua Qingxue’ cuttings. (A–C) represent rates of rooting/callus/rot, root number, and root length of different polyamines on the rooting of ‘Jinghua Qingxue’ cuttings, respectively. The cuttings were initially dipped in the solutions of Put, Spd, and Spm at three concentrations (0.05 mM, 0.1 mM, and 1.0 mM) for five mins and then dipped in IBA solution (2000 mg·L−1) for 15 min with water as a control. Different letters indicate significant differences at the p level below 0.05.
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Figure 3. Effect of substrates on rooting parameters of ‘Jinghua Qingxue’ cuttings. (A–C) represent rates of rooting/callus/rot, root number, and root length of different substrates on rooting of ‘Jinghua Qingxue’ cuttings, respectively. T1, T2, T3, T4, T5, T6, and T7 represent cocopeat (100%), cocopeat/perlite (v:v 1:1), cocopeat/perlite (v:v 1:2), perlite/vermiculite (v:v 7:3), perlite/peat (v:v 3:1), perlite/peat (v:v 4:6), and perlite/peat/vermiculite (v:v:v 3:1:1), respectively. The cuttings were treated with IBA 2000 mg·L−1 for 15 min and inserted into the seven substrates. Different letters indicate significant differences at the p level below 0.05.
Figure 3. Effect of substrates on rooting parameters of ‘Jinghua Qingxue’ cuttings. (A–C) represent rates of rooting/callus/rot, root number, and root length of different substrates on rooting of ‘Jinghua Qingxue’ cuttings, respectively. T1, T2, T3, T4, T5, T6, and T7 represent cocopeat (100%), cocopeat/perlite (v:v 1:1), cocopeat/perlite (v:v 1:2), perlite/vermiculite (v:v 7:3), perlite/peat (v:v 3:1), perlite/peat (v:v 4:6), and perlite/peat/vermiculite (v:v:v 3:1:1), respectively. The cuttings were treated with IBA 2000 mg·L−1 for 15 min and inserted into the seven substrates. Different letters indicate significant differences at the p level below 0.05.
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Figure 4. The morphological and anatomical structure of shoot section at different phenological stages in the tree peony ‘HN’. (A–D) represent the status of cuttings at 10 DBF, 28 DAF, 55 DAF (2022), and 80 DAF in 2021, respectively. DBF: day before flowering stage, DAF: day after flowering stage. (a–d) indicated the anatomical structure of the stem from 10 DBF to 80 DAF. The flowering stages were on 15 May 2021 and 7 May 2022, respectively. Abbreviations: Ep, epidermis; Co, cortex; Scl, sclerenchyma; Ph, phloem; Xy, xylem. (a–d): bar = 100 μm.
Figure 4. The morphological and anatomical structure of shoot section at different phenological stages in the tree peony ‘HN’. (A–D) represent the status of cuttings at 10 DBF, 28 DAF, 55 DAF (2022), and 80 DAF in 2021, respectively. DBF: day before flowering stage, DAF: day after flowering stage. (a–d) indicated the anatomical structure of the stem from 10 DBF to 80 DAF. The flowering stages were on 15 May 2021 and 7 May 2022, respectively. Abbreviations: Ep, epidermis; Co, cortex; Scl, sclerenchyma; Ph, phloem; Xy, xylem. (a–d): bar = 100 μm.
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Figure 5. The adventitious root formation of softwood cuttings of the tree peony ‘High Noon’. (A) The cutting status on 0 DAC. (B) Leaves turned green on 28 DAC. (C) Leaves unfolding on 56 DAC. (D) Adventitious root formation at 62 DAC. (E) The callus at the base incision at 10 DAC. (F) Callus tissues enlarging at 22 DAC. (G) Nodules formed from callus at 56 DAC. (H,I) ARs out of the callus or bark at 62 DAC. DAC: days after cutting.
Figure 5. The adventitious root formation of softwood cuttings of the tree peony ‘High Noon’. (A) The cutting status on 0 DAC. (B) Leaves turned green on 28 DAC. (C) Leaves unfolding on 56 DAC. (D) Adventitious root formation at 62 DAC. (E) The callus at the base incision at 10 DAC. (F) Callus tissues enlarging at 22 DAC. (G) Nodules formed from callus at 56 DAC. (H,I) ARs out of the callus or bark at 62 DAC. DAC: days after cutting.
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Figure 6. The rooting type of softwood cuttings of tree peony. (A) Callus induction; (B) bark-derived rooting; (C) integrated root induction; (D) the status of cutting at 0 DAC; (E) the section observation of AR from the callus; (F) the section observation of AR out of the vascular cambium. Abbreviations: Ca, callus tissue; AR, adventitious root; Ep, epidermis; Co, cortex; Ph, phloem; Vc, vascular; Xy, xylem.
Figure 6. The rooting type of softwood cuttings of tree peony. (A) Callus induction; (B) bark-derived rooting; (C) integrated root induction; (D) the status of cutting at 0 DAC; (E) the section observation of AR from the callus; (F) the section observation of AR out of the vascular cambium. Abbreviations: Ca, callus tissue; AR, adventitious root; Ep, epidermis; Co, cortex; Ph, phloem; Vc, vascular; Xy, xylem.
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Figure 7. The propagation protocol of softwood cuttings in the tree peony ‘High Noon’.
Figure 7. The propagation protocol of softwood cuttings in the tree peony ‘High Noon’.
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Table 1. The treatment of plant growth regulators on rooting in the tree peony ‘High Noon’.
Table 1. The treatment of plant growth regulators on rooting in the tree peony ‘High Noon’.
Treatment CodePlant Growth RegulatorConcentration
(mg·L−1/Dilution Factor)		Soaking Time
(min/s)
IBA-1000IBA100015
IBA-2000IBA200015
IBA-3000IBA300015
GP-150GenPan15015
GP-250GenPan25015
GP-350GenPan35015
RPRooting Powder0.25% IBA3 s
CKWater-15
Table 2. The treatment of polyamines on rooting in the tree peony ‘Jinghua Qingxue’.
Table 2. The treatment of polyamines on rooting in the tree peony ‘Jinghua Qingxue’.
Treatment CodePolyamineConcentration
(mM)		Soaking Time
(min)
Put-0.05Put0.055
Put-0.1Put0.15
Put-1.0Put1.05
Spd-0.05Spd0.055
Spd-0.1Spd0.15
Spd-1.0Spd1.05
Spm-0.05Spm0.055
Spm-0.1Spm0.15
Spm-1.0Spm1.05
CKWater-5
Table 3. The effect of cutting phenological stage on rooting in tree peony.
Table 3. The effect of cutting phenological stage on rooting in tree peony.
YearCultivarDBF/DAFRooting Rate (%)Callus Rate (%)Rot Rate (%)
2021HN10DBF38.33 ± 10.27 ab100.00 a6.67 ± 4.71 j
28DAF50.00 ± 8.16 a90 ± 8.16 ab33.33 ± 4.71 g
80DAF6.67 ± 4.71 c40 ± 8.16 e70 ± 8.16 d
JHQX10DBF31.67 ± 6.24 ab100.00 a50 ± 8.16 f
29DAF13.33 ± 4.17 bc20 ± 8.16 h70 ± 8.16 d
80DAF0.00 c16.67 ± 4.71 hi73.33 ± 9.43 d
JYG10DBF21.67 ± 8.5076.67 ± 12.47 c63.33 ± 4.71 e
29DAF6.67 ± 4.71 c13.33 ± 4.71 j81.67 ± 6.24 bc
81DAF0.00 c6.67 ± 4.71 j93.33 ± 4.71 ab
GYSH10DBF16.67 ± 9.43 bc80 ± 8.16 d66.67 ± 4.71 de
30DAF6.67 ± 4.71 c13.33 ± 4.71 j83.33 ± 4.71 bc
81DAF06.67 ± 4.71 j96.67 ± 4.71 a
2022HN18DAF16.67 ± 4.71 bc96.67 ± 4.71 a10.00 ± 8.16 i
35DAF10.00 ± 8.16 c93.33 ± 4.71 bc23.33 ± 4.71 h
55DAF6.67 ± 4.71 c40.00 ± 8.16 fg70.00 ± 8.16 d
70DAF0.00 c21.67 ± 8.50 gh83.33 ± 4.71 bc
BZL36DAF30.00 ± 8.16 ab100.00 a13.33 ± 4.71 i
58DAF6.67 ± 4.71 c80.00 ± 8.16 c23.33 ± 12.47 h
72DAF0.00 c16.67 ± 9.43 j80.00 ± 8.16 bc
JHQX19DAF53.33 ± 12.47 a96.67 ± 4.71 ab6.67 ± 4.71 j
36DAF7.50 ± 5.40 c85.83 ± 4.25 bc25.00 ± 4.08 h
56DAF3.33 ± 4.71 c39.17 ± 8.25 fg71.67 ± 2.36 d
GYSH20DAF6.67 ± 4.71 c46.67 ± 4.71 e66.67 ± 9.43 de
38DAF3.33 ± 4.71 c25.00 ± 4.08 gh75.00 ± 4.08 cd
58DAF0.00 c10.00 ± 8.16 ij80.00 ± 8.16 bc
JYG10DAF0.00 c10.00 ± 8.16 ij86.67 ± 4.71 ab
22DAF10.83 ± 1.18 c35.83 ± 4.25 ef78.33 ± 2.36 cd
37DAF0.00 c6.67 ± 4.71 j85.83 ± 4.25 b
JZM10DAF0.00 c17.50 ± 3.54 hi78.33 ± 2.36 cd
45DAF6.67 ± 4.71 c29.17 ± 7.17 fg70.83 ± 7.17 d
68DAF0.00 c3.33 ± 4.71 k83.33 ± 4.71 bc
JH40DAF3.33 ± 4.71 c21.67 ± 2.36 hi85.83 ± 4.25 b
62DAF0.00 c3.33 ± 4.71 k93.33 ± 4.71 ab
JHL41DAF6.67 ± 4.71 c20.83 ± 7.16 h89.17 ± 1.18 b
63DAF0.00 c3.33 ± 4.71 k96.67 ± 4.71 a
LYH44DAF3.33 ± 4.71 c20.00 ± 8.16 h75.00 ± 4.08 d
65DAF0.00 c3.33 ± 4.71 k93.33 ± 4.71 ab
Note: The abbreviations of the nine cultivars are BZL, HN, JHQX, JYG, JH, JZM, JHL, GYSH, and LYH for ‘Bartzella’, ‘High Noon’, ‘Jinghua Qingxue’, ‘Jing Yunguan’, ‘Jinghong’, ‘Jing Zuimei’, ‘Jing Helan’, ‘Gaoyuan Shenghuo’, and ‘Luoyang Hong’, respectively. The flowering stages were on the 15 May 2021 and 7 May 2022, respectively. DBF: day before flowering stage, DAF: day after flowering stage. The cuttings were dipped in IBA solution (2000 mg·L−1) for 15 min and inserted in peat/perlite substrate (v:v 6:4). Different letters in the same column indicate significant differences at the p level below 0.05.
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MDPI and ACS Style

Li, D.; Cheng, F.; Tao, X.; Zhong, Y. Effect of Cutting Phenological Stage, Chemical Treatments, and Substrate on Rooting Softwood Cuttings of Tree Peony. Horticulturae 2025, 11, 552. https://doi.org/10.3390/horticulturae11050552

AMA Style

Li D, Cheng F, Tao X, Zhong Y. Effect of Cutting Phenological Stage, Chemical Treatments, and Substrate on Rooting Softwood Cuttings of Tree Peony. Horticulturae. 2025; 11(5):552. https://doi.org/10.3390/horticulturae11050552

Chicago/Turabian Style

Li, Dongli, Fangyun Cheng, Xiwen Tao, and Yuan Zhong. 2025. "Effect of Cutting Phenological Stage, Chemical Treatments, and Substrate on Rooting Softwood Cuttings of Tree Peony" Horticulturae 11, no. 5: 552. https://doi.org/10.3390/horticulturae11050552

APA Style

Li, D., Cheng, F., Tao, X., & Zhong, Y. (2025). Effect of Cutting Phenological Stage, Chemical Treatments, and Substrate on Rooting Softwood Cuttings of Tree Peony. Horticulturae, 11(5), 552. https://doi.org/10.3390/horticulturae11050552

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