Optimizing Transplanting Practices for Potted Tree Peony Based on Non-Structural Carbohydrates Accumulation

Abstract

Potted cultivation serves as a vital strategy for industrialized production of standardized tree peonies, engineering seedlings capable of year-round and off-site transplantation. However, the limited root zone in potted conditions restricts root development, resulting in suboptimal seedling quality and hindering commercial-scale production. This study aimed to investigate the relationship between the accumulation characteristics of non-structural carbohydrates (NSCs) and growth performance in potted tree peonies, while also optimizing the transplantation technologies for potted cultivation. Using two-year-old grafted seedlings of ‘Luoyanghong’ as experimental material, the effects of root pruning, rooting agent, and Metarhizium anisopliae application on morphological development and NSCs accumulation in potted tree peony seedlings were investigated. The results showed that old roots serve as the primary storage organs for NSCs in the potted tree peony. Slight root pruning (25%) was beneficial for fibrous root growth, whereas excessive root pruning (50%) resulted in reduced biomass and NSCs accumulation. The application of a high concentration of rooting agents effectively promoted root growth and mitigated the adverse effects of root pruning. Furthermore, Metarhizium anisopliae significantly increased the stem number in potted tree peonies. The optimal protocol identified through range analysis involved 25% root pruning, followed by irrigation with a solution containing 750 mg·L⁻¹ rooting agent and 20 million spores·mL⁻¹ of Metarhizium anisopliae. The rational distribution of NSCs and coordinated growth across different organs enhanced NSCs accumulation in potted tree peonies. These results demonstrate that combining root pruning with the application of rooting agent and Metarhizium anisopliae can effectively increase NSCs accumulation, optimize plant morphology, and ultimately improve the quality of potted tree peony seedlings.

1. Introduction

Tree peony (Paeonia suffruticosa Andr.) is a valuable flower that originated in China, renowned for its vibrant colors and intricate patterns [1]. It has traditionally been used as a field ornamental since ancient times. However, with the increasing demand for ornamental varieties and advancements in cultivation techniques, potted tree peonies have gradually become an important focus of interest [2,3]. Although conventional field cultivation remains the predominant production method owing to its relatively low maintenance cost and simple management requirements [4], potted cultivation has gained increasing popularity in off-site and off-season potted flower production because of its flexibility and ease of management in facilities [5,6]. However, compared to traditional field production, potted tree peonies experience significantly more constrained growth conditions, which currently limit their commercial-scale adoption.

In potted cultivation, tree peony seedlings are transplanted from the field into containers for intensive management, enabling the plant to flower once or multiple times [7]. Due to the large fleshy root system of tree peonies and the limited space of the pot, the thick roots must be coiled and folded within the pots, which hinders the growth of lateral roots and nutrient absorption and transportation, resulting in variations in nutrient supply to stems and subsequently uneven growth of the stem [8,9]. Before potting, tree peonies typically undergo root pruning to adapt the roots to the relatively restricted conditions of pot culture. Proper root pruning effectively inhibits the growth of primary roots while stimulating lateral root development, thereby increasing root density, optimizing root architecture, and collectively enhancing seedling quality [10]. However, excessive root pruning leads to nutrient depletion in roots and adversely affects plant growth [11]. Therefore, it is important to explore the optimal degree of root pruning for potted tree peonies. Additionally, prolonged exposure to dry air during seedling transplantation can cause severe damage to the delicate fibrous roots, often leading to delayed seedling growth, reduced transplanting success rates, and even plant death [12]. Therefore, promoting the formation of fibrous roots is necessary for transplanting. Previous studies have shown that rooting agents, containing phytohormones, have been widely used to increase rooting, which not only improves the survival rate of plants but also plays a significant role in improving the quality of potted tree peony flowers [13,14]. In addition to promoting root growth, rooting agents can improve plant resilience by regulating hormone levels and metabolic processes [15]. While the optimal concentration of root agents is influenced by their composition and crop species, the optimal concentration of its application in conjunction with other treatments has yet to be determined experimentally. Tree peony seedlings exhibit high susceptibility to root-feeding insect pests in nursery conditions, notably scarab beetles, which directly damage the fleshy root system, compromising plant vigor and survival rates [16]. In traditional production, the use of highly toxic chemical pesticides is the primary method to eliminate pests. Although this method is effective, it causes harmful side effects that damage the environment [17]. With increasing environmental awareness among consumers, pesticide-treated potted plants face growing market rejection due to ecological concerns. Therefore, using highly specific, non-polluting biological insecticides for pest control represents an important approach for improvement in the industry. Previous studies have shown that Metarhizium anisopliae not only has specific pathogenicity against scarab larvae and persistently colonizes in soil, providing long-term protection for plants [18], but can also endophytically colonize plant roots, thereby enhancing plant growth [19,20]. However, how to effectively combine these treatments remains a subject worthy of experimental research.

During the growing season, deciduous tree species accumulate substantial amounts of NSCs through photosynthetic assimilation. These NSCs serve as a carbon source supporting respiration, cold resistance, and signaling during winter. They also provide the materials and energy required for bud break and shoot elongation in spring [21,22]. Tree peonies, as deciduous shrubs, exhibit winter dormancy. In winter, portions of the stems and all leaves senesce and abscise. The following spring, the new stems, leaves and flowers regenerate, requiring substantial NSCs reserves [23,24]. Sufficient NSCs reserves are critical for high flower quality in the tree peony [25]; however, patterns of NSCs accumulation in potted tree peonies and their physiological roles during early development remain poorly understood. Furthermore, the potential synergistic effects of these treatments on NSCs distribution and subsequent growth performance require systematic investigation.

Transplanting potted tree peonies entails a series of coordinated cultivation measures to ensure robust seedling growth. To efficiently integrate these measures, we used an L9(3⁴) orthogonal array design with three treatment levels of root pruning, rooting agent, and Metarhizium anisopliae. The goal was to identify the optimal combination that significantly enhances seedling NSCs accumulation, thereby providing essential data and a theoretical basis for the advancement of commercial production of tree peonies.

2. Materials and Methods

2.1. Experimental Site

The experimental site is located at the potting base of Henan University of Science and Technology, Luoyang City, Henan Province (Latitude 34°47′18″ N, longitude112°34′40″ E, elevation 191 m), characterized by a warm-temperate continental monsoon climate. The potting substrate was composed of grass charcoal soil mixed with rotted organic fertilizer. The substrate had a bulk density of 0.3 g·cm⁻³, pH 6.7, and contained 400 mg·g⁻¹ organic matter, 230 mg·g⁻¹ total carbon, 15 mg·g⁻¹ total nitrogen, 500 µg·g⁻¹ available phosphorus, and 30 µg·g⁻¹ available potassium [26].

2.2. Experimental Material

In October 2020, uniform-sized, pest- and disease-free 2-year-old ‘Luoyanghong’ grafted seedlings were selected. These seedlings were randomly divided into nine groups after being cut back to the ground. The experiment employed a three-factor, three-level orthogonal design (L9(3⁴)), resulting in nine combinations. Factor A (root pruning treatment) involved removing 25%, 33%, and 50% of the old roots from the root tips (slight, medium, and severe pruning, respectively). Factor B (rooting agent treatment) involved irrigating with rooting agents, our laboratory’s patented technology [27], at concentrations of 250, 500, and 750 mg·L⁻¹ (low, medium, and high concentrations, respectively) of tree peony transplant rooting agent. Factor C (Metarhizium anisopliae treatment) involved irrigating with biological insecticide Metarhizium anisopliae (Greenation, Chongqing, China) at concentrations of 10 million, 15 million, and 20 million spores·mL⁻¹ (low, medium, and high concentrations, respectively), with 500 mL per pot. The experimental design is shown in

Table 1. Orthogonal test design of potted tree peony experiment.

Combinations	Factors
	Root Pruning (A) /%	Rooting Agent (B) /(mg·L−1)	Metarhizium anisopliae (C) /(Million Spores·mL−1)
1	25	250	10
2	25	500	15
3	25	750	20
4	33	250	15
5	33	500	20
6	33	750	10
7	50	250	15
8	50	500	10
9	50	750	20

Note: A—Root pruning treatment; B—Rooting agent treatment; C—Metarhizium anisopliae treatment.

. The cultivation container was a black plastic pot measuring 25 cm in height and 22 cm in inner diameter, filled with 5 kg of substrate. Forty single-plant replicates were prepared for each combination, and the pots were maintained under conventional water and fertilizer management modes. The seedlings were sampled in mid-April of the following year to investigate growth performance of tree peonies under different combinations.

2.3. Measurement of Photosynthetic Performance

In mid-April 2021, after the tree peony leaves were fully expanded, the photosynthetic performance of the leaves, including net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Cs), and intercellular carbon dioxide concentration (Ci), was measured under a fixed light intensity of 1000 µmol/(m²·s) using a Li-6400XT portable photosynthesis system (Li-COR, Tucson, AZ, USA), and the water use efficiency (WUE) was calculated as the ratio of Pn/Tr. The SPAD value of the top leaves was determined using a SPAD-502 Plus Chlorophyll Meter (Konica Minolta Sensing, Osaka, Japan). Three representative plants were selected for each combination, with a total of 40 plants per combination, and three leaves were measured per plant.

2.4. Measurement of Morphological Indicators

Plant morphological indicators were assessed by selecting three representative plants from each combination, measuring stem number and stem length, followed by washing the roots with tap water. Seedlings were then separated into different organs, including leaves, stems, old roots, and fibrous roots. The fresh biomass of these organs was determined using an electronic balance. The root-to-shoot ratio was calculated as the combined mass of old roots divided by the whole plant biomasss. Leaf area was analyzed using Digimizer image analysis software (MedCalc Software Ltd., Ostend, Belgium). Some of the samples were snap-frozen in liquid nitrogen and subsequently stored at −40 °C for the determination of physiological and biochemical indices.

2.5. Measurement of NSCs

The soluble sugar and starch contents of each organ of the tree peony were determined using the sulfuric acid–anthracene ketone method [28]. Non-structural carbohydrates (NSC) content was calculated as the sum of soluble sugar and starch content. NSC accumulation (NSCA) in each organ was obtained by multiplying the organ biomass by its NSCs content, while the total non-structural carbohydrate accumulation (TNSCA) of each plant was calculated as the sum of NSCs across all organs.

2.6. Statistical Analysis

Data from the experiments were organized using Excel 2019 (Microsoft Crop, Redmond, WA, USA), and multiple comparisons, correlation analysis, range analysis, and path analysis were performed on SPSS 17.0. (SPSS Inc., Chicago, IL, USA). Graphs were created using Origin 2025 (OriginLab Corporation, Northampton, MA, USA).

3. Results

3.1. Changes in Morphological Characteristics of Potted Tree Peony

There are significant differences in the morphological characteristics of different treatments in potted tree peonies. Under slight and medium root pruning conditions, the number of stems increased as the concentration of Metarhizium anisopliae increased, whereas severe root pruning significantly reduced the stem length of potted tree peonies (Figure S1). Comparing the effects of different treatments on biomass revealed that root pruning had a negative effect on tree peony’s biomass, with the biomass of leaves, old roots, fibrous roots, and the whole plant, as well as the ratio of root to seedling, showing a decreasing trend as the pruning intensity increased. However, high concentrations of rooting agent promoted biomass accumulation in leaves, fibrous roots and the whole plant (Figure 1). The analysis of biomass distribution among different organs revealed that the biomass of old roots was the highest, followed by leaves and stems, with fibrous roots being the lowest. However, with the accumulation of photosynthetic products and the expansion of new roots, this distribution pattern may change. Overall, combination 3 was identified as the most effective, producing the highest stem number and the greatest biomass of leaves, old roots, fibrous roots and the whole plant.

3.2. Changes in Photosynthetic Performance of Potted Tree Peonies

Photosynthesis represents the predominant pathway for carbohydrate assimilation in plants. The parameters of leaf photosynthetic performance (Pn, Gs, Ci, Tr and WUE), LA and SPAD values of potted tree peonies were measured (

Table 2. Changes in photosynthetic performance of potted tree peony leaves.

Combinations	Pn /(µmolCO2 ·m−2s−1)	Gs /(molH2O ·m−2s−1)	Ci /(µmol· mol−1)	Tr /(mmolH2O ·m−2s−1)	WUE /(µmolCO2 mmol−1H2O)	LA /cm2	SPAD Valve
1	4.82 ± 0.70 abc	0.04 ± 0.01	193.62 ± 31.63	1.05 ± 0.27 ab	4.87 ± 1.44 a	1554.1 ± 95.1 abc	57.30 ± 2.27 d
2	4.52 ± 0.65 abc	0.04 ± 0.01	206.00 ± 21.60	1.08 ± 0.14 ab	4.25 ± 0.84 abc	1192.2 ± 80.8 de	58.21 ± 3.17 bcd
3	5.40 ± 0.70 a	0.04 ± 0.01	195.09 ± 13.24	1.23 ± 0.20 a	4.48 ± 0.74 ab	1792.3 ± 256.8 a	60.34 ± 2.96 a
4	5.16 ± 1.23 ab	0.04 ± 0.01	214.14 ± 20.77	1.26 ± 0.21 a	4.14 ± 0.85 abc	1269.9 ± 299 cde	59.49 ± 2.14 abc
5	4.94 ± 1.15 abc	0.04 ± 0.01	199.34 ± 14.28	1.14 ± 0.13 ab	4.34 ± 0.86 abc	1237.0 ± 49.6 de	59.97 ± 2.91 ab
6	3.41 ± 0.79 d	0.03 ± 0.01	198.13 ± 27.59	0.92 ± 0.28 b	3.89 ± 0.78 abc	1434.7 ± 249.2 bcd	59.63 ± 2.52 ab
7	3.99 ± 1.10 cd	0.03 ± 0.01	199.90 ± 21.95	1.05 ± 0.19 ab	3.76 ± 0.52 c	1028.9 ± 137.5 e	58.06 ± 2.24 bcd
8	4.28 ± 0.57 bcd	0.04 ± 0.01	209.29 ± 26.15	1.22 ± 0.24 a	3.55 ± 0.36 c	1097.5 ± 192.2 e	58.29 ± 2.38 bcd
9	4.20 ± 0.70 bcd	0.03 ± 0.01	187.25 ± 34.98	1.10 ± 0.16 ab	3.84 ± 0.63 bc	1213.2 ± 203.7 de	57.64 ± 3.83 cd

Note: Pn—Net photosynthetic rate; G_S—Stomatal conductance; Ci—Intercellular CO₂ concentration; Tr—Transpiration rate; WUE—Water use efficiency; LA—Leaf area per plant. The data are shown as means ± SE (n = 3). Different lowercase letters represent statistically significant differences among the various indicators (p < 0.05).

), and the results showed that, except for treatment 6, which exhibited lower Pn and Tr values, the differences in Pn, Gs, Ci, and Tr among the treatments were relatively small. However, WUE and LA showed a decreasing trend with increasing root pruning intensity, while a high concentration of rooting agent promoted LA. These results indicate that the rooting agent can mitigate the negative effects of nutrient loss on leaf physiological performance.

3.3. Accumulation and Distribution of NSCs in Different Organs of Potted Tree Peonies

Soluble sugar is an important form of NSCs for storage and transportation. A comparison of the soluble sugar content in different organs of the tree peony revealed that the leaves and old roots, the primary organs for the production and storage of photosynthetic products, contained higher levels of soluble sugar than the other organs (

Table 3. Soluble sugar content in different organs of tree peony /mg·g⁻¹.

Combinations	Leaf	Stem	Old Root	Fibrous Root
1	68.98 ± 4.47 a	43.24 ± 1.08 bcd	55.35 ± 2.57 bcd	21.66 ± 3.43 abcd
2	69.48 ± 4.11 a	56.56 ± 1.28 a	60.87 ± 4.49 abc	26.61 ± 3.28 ab
3	60.21 ± 4.61 bc	46.61 ± 3.14 b	68.82 ± 4.25 a	26.85 ± 3.51 a
4	58.52 ± 1.46 c	40.93 ± 1.74 cd	63.99 ± 8.82 ab	18.01 ± 2.72 d
5	58.50 ± 1.33 c	45.04 ± 0.41 bc	58.44 ± 2.13 bc	20.98 ± 0.64 bcd
6	60.62 ± 3.21 bc	41.34 ± 3.23 cd	53.35 ± 0.71 cd	23.97 ± 2.88 abc
7	59.94 ± 3.88 bc	39.34 ± 0.78 de	63.52 ± 2.68 ab	23.14 ± 1.03 abcd
8	57.08 ± 2.82 c	39.98 ± 3.70 de	47.23 ± 2.63 d	17.55 ± 0.99 d
9	59.76 ± 0.52 bc	39.90 ± 1.94 de	47.69 ± 3.77 d	20.38 ± 0.96 cd

Note: The data are shown as means ± SE (n = 3). Different lowercase letters represent statistically significant differences among the various indicators (p < 0.05).

). In contrast, fibrous roots, serving as the main absorption organs, contained lower soluble sugar levels. Stems, which act as the main channels for the transport of substances between above- and below-ground parts, had intermediate soluble sugar levels. Thus, there is a significant concentration gradient of soluble sugar among different organs in the potted tree peony. Analysis of the effects of different treatments on soluble sugar content showed that the soluble sugar content in the stems decreased with the increase of root pruning. Slight root pruning increased soluble sugar content in the leaves, indicating that the soluble sugar content is primarily influenced by organ type and the degree of root pruning.

Starch is an important storage form of NSCs. Old roots, the main storage organ of the tree peony, contained the highest starch contents, followed by leaves and stems, while fibrous roots had the lowest (

Table 4. Starch content in different organs of potted tree peony /mg·g⁻¹.

Combinations	Leaf	Stem	Old Root	Fibrous Root
1	18.07 ± 2.96 ab	21.96 ± 1.82 ab	71.44 ± 8.68 cd	8.25 ± 1.39 c
2	18.90 ± 0.84 ab	17.69 ± 1.95 d	47.35 ± 8.17 e	7.79 ± 1.28 c
3	20.91 ± 1.37 ab	20.92 ± 0.84 bc	98.28 ± 11.41 a	10.01 ± 0.80 abc
4	21.35 ± 1.80 ab	21.79 ± 0.53 ab	83.69 ± 4.41 bc	8.23 ± 1.17 c
5	20.76 ± 2.14 ab	18.19 ± 2.64 cd	66.36 ± 6.18 d	9.22 ± 0.62 bc
6	17.01 ± 1.49 b	22.68 ± 1.03 ab	68.64 ± 8.01 d	8.70 ± 0.69 c
7	18.83 ± 2.20 ab	22.63 ± 0.65 ab	77.31 ± 2.65 bcd	11.01 ± 1.09 ab
8	21.49 ± 1.14 ab	16.89 ± 1.36 d	52.27 ± 0.82 e	7.74 ± 1.56 c
9	18.51 ± 4.49 ab	19.56 ± 1.90 bcd	73.81 ± 5.06 bcd	9.22 ± 0.49 bc

Note: The data are shown as means ± SE (n = 3). Different lowercase letters represent statistically significant differences among the various indicators (p < 0.05).

). This concentration gradient centered in the old roots confirms their role as primary starch reservoir. However, the effects of different treatments on starch content in tree peony organs were not statistically significant.

NSCs content is widely recognized as a powerful indicator of plant nutrient status. The results showed that NSCs content was highest in old roots, followed by leaves and stems, while fibrous roots had the lowest values, confirming that old roots are the most important NSCs storage organs in the tree peony (

Table 5. NSCs content in different organs of potted tree peony /mg·g⁻¹.

Combinations	Leaf	Stem	Old Root	Fibrous Root
1	94.99 ± 5.85 a	65.20 ± 2.46 bc	126.79 ± 6.44 cd	29.91 ± 4.39 bcde
2	96.21 ± 4.63 a	74.26 ± 2.81 a	108.21 ± 12.65 ef	34.41 ± 2.08 ab
3	79.2 ± 4.81 b	67.53 ± 2.99 b	167.1 ± 12.6 a	36.86 ± 4.14 a
4	81.69 ± 1.52 b	62.72 ± 1.23 bcd	147.69 ± 13.23 b	26.24 ± 2.37 de
5	81.14 ± 0.57 b	63.24 ± 2.23 bcd	124.8 ± 7.85 cde	30.2 ± 1.26 bcd
6	82.25 ± 5.42 b	64.02 ± 3.84 bcd	121.99 ± 8.28 de	32.67 ± 2.47 abc
7	78.3 ± 4.5 b	61.97 ± 0.74 cd	140.83 ± 3.58 bc	34.14 ± 1.83 abc
8	77.94 ± 3.6 b	56.87 ± 4.49 e	99.5 ± 2.52 f	25.29 ± 2.54 e
9	82.16 ± 2.47 b	59.46 ± 2.76 de	121.5 ± 7.08 de	29.6 ± 1.37 cde

Note: The data are shown as means ± SE (n = 3). Different lowercase letters represent statistically significant differences among the various indicators (p < 0.05).

). With the increase of root pruning, NSCs content in leaves and stems displayed a decreasing trend, indicating that root pruning significantly affected the aboveground nutrient status of potted tree peonies.

NSCs accumulation (NSCA) is widely used to assess the nutrient accumulation and seedling quality. In terms of NSCs accumulation across different organs, the accumulation order from highest to lowest was old roots > leaves > stems > fibrous roots (Figure 2). The old roots accounted for approximately 60% of the total NSCs, further confirming their roles as the primary storage organ in the tree peony. Regarding treatment effects, NSCs accumulation in leaves, old roots, fibrous roots, and the whole plant tended to decrease with increasing root pruning intensity. Under slight and medium root pruning conditions, high concentrations of rooting agent promoted NSCs accumulation in old roots, fibrous roots, and the whole plant. However, the effect of Metarhizium anisopliae on NSCs accumulation remains unclear and requires further investigation.

3.4. Range Analysis of NSCs Accumulation in Potted Tree Peony

The results of range analysis showed that the effects of each factor on TNSCA accumulation were in the order of root pruning > rooting agent > Metarhizium anisopliae (

Table 6. Range analysis of NSCs accumulation in tree peony seedlings.

Combinations	Root Pruning (A) /%	Rooting Agent (B) /(mg·L−1)	Metarhizium anisopliae (C) /(Million Spores·mL−1)	TNSCA /(g·Plant−1)
1	25	250	10	15.15
2	25	500	15	14.39
3	25	750	20	22.86
4	33	250	15	13.60
5	33	500	20	12.18
6	33	750	10	14.71
7	50	250	15	11.22
8	50	500	10	9.20
9	50	750	20	10.62
k1	17.4668	13.3246	13.0209
k2	13.4978	11.9233	12.8696
k3	10.3471	16.0638	15.4212
Range	7.1197	4.1405	2.5516
Better level	A1	B3	C3
Factor priority	A > B > C
Optimal combination	A1B3C3

Note: A—Root pruning treatment; B—Rooting agent treatment; C—Metarhizium anisopliae treatment.

). The optimal combination was treatment 3, which involved pruning 25% of the old roots and irrigating with 500 mL of a mixed solution containing 750 mg·L⁻¹ rooting agent and 20 million spores·mL⁻¹ of Metarhizium anisopliae. These results indicate that slight root pruning combined with high concentrations of rooting agent and Metarhizium anisopliae is effective in promoting NSC accumulation in tree peonies.

3.5. Correlation of Photosynthetic Performance and Morphological Characteristics with NSCs Accumulation in Potted Tree Peony

Correlation analysis was used to investigate the relationship of these indices (Figure 3). The results showed that stem number and stem length were significantly or highly significantly correlated with old root, fibrous root, whole plant biomass, and root-to-shoot ratio. There were significant and highly significant positive correlations between fibrous root, leaf, old root and whole plant biomass, but only leaf significantly and positively correlated with stem. The relationship between photosynthetic performance and morphology indicated that Pn was highly significantly and positively correlated with stem number, leaf biomass, and fibrous root biomass. In contrast, Tr and Ci showed highly significant negative correlations with stem biomass. LA is significantly positively correlated with the biomass of all organs. The TNSCA was significantly and positively correlated with stem number, stem length, leaf biomass, old root biomass, and fibrous root biomass, as well as with WUE, LA, SPAD, NSCs content in stems, old roots, and fibrous roots, and NSCA accumulation in each organ. These results indicate that TNSCA plays an important role in the growth of potted tree peonies.

Furthermore, stepwise regression analysis was performed with TNSCA as the dependent variable and other indices involved in morphology, photosynthesis and NSCs content as the independent variables, yielding the regression equation Y = −8.334 + 0.057 × WM + 0.083 × ORNSC + 0.395 × R/S − 0.099 × Ci + 0.245 × FM − 0.12 × FRNSC, with R² = 0.962. The strong predictive capacity of this model suggests that these six parameters are key determinants influencing whole plant NSCs accumulation. The ranking of their direct path coefficients to TNSCA is ORNSC > R/S > WM > FM > FRNSC > Ci (

Table 7. Path analysis of the key factors affecting NSCs accumulation in whole plant.

Factors	Direct Path Coefficient	Indirect Path Coefficient
		→WM	→ORNSC	→R/S	→Ci	→FM	→FRNSC	Total Path Coefficient
WM	0.379		0.164	0.208	0.043	0.128	−0.077	0.844
ORNSC	0.452	0.137		0.041	0.028	0.083	−0.039	0.702
R/S	0.401	0.196	0.046		−0.068	0.033	−0.057	0.551
Ci	−0.204	−0.080	−0.061	0.134		−0.043	0.048	−0.205
FM	0.184	0.263	0.205	0.071	0.047		−0.053	0.718
FRNSC	−0.134	0.219	0.130	0.169	0.073	0.072		0.529

Note: The columns marked with → represent indirect path coefficients.

). However, the total path coefficients followed a different order: WM > FM > ORNSC > R/S > FRNSC > Ci. This indicates that WM exerted stronger influences on TNSCA through both direct and indirect pathways. Although the direct path coefficient between FRNSC and TNSCA is negative, the indirect path coefficient of FRNSC on TNSCA is positive. This may be related to the fact that during fibrous roots growth, plants increase their input of NSCs, which promotes the growth of fibrous roots and enhances their capacity to absorb water and nutrients, thereby facilitating the accumulation of NSCs. This indicates that transporting NSCs to fibrous roots and promoting their growth represents an important survival strategy for potted tree peonies. The direct correlation coefficient between ORNSC and TNSCA is the highest, indicating that NSCs in old roots have the greatest direct impact on TNSCA. The path coefficient of Ci with TNSCA is negative, suggesting that higher CO₂ utilization efficiency in leaves is beneficial for NSCs accumulation. Therefore, NSCs accumulation in potted tree peonies is regulated through an integrated physiological process involving plant morphology, root NSCs concentration, and photosynthesis. Consequently, TNSCA represents a robust integrative physiological indicator that effectively reflects the seedling growth status.

4. Discussion

4.1. The Effects of Root Pruning, Rooting Agents and Metarhizium Anisopliae

During the process of tree peony transplanting, proper root pruning facilitates root morphological modifications that enhance container adaptability [29], thereby improving seedling uniformity. However, excessive root pruning may result in nutrient depletion and adversely affect plant growth. Our results showed that under slight root pruning conditions, the LA, biomass of leaf, old root and fibrous root, and NSCs accumulation of potted tree peonies achieved the highest levels. These values decreased with increasing pruning intensity, indicating that root pruning caused nutrient loss from roots and had a negative effect on seedling growth. Rooting agents, which usually consist of multiple nutrients and plant growth regulators, can promote the growth and development of fibrous roots and accelerate the completion of root morphology, so they are widely used in seedling transplanting and cuttings [30,31,32]. Previous studies have indicated that the application of rooting agents improves the survival rate, height, and diameter of Larch seedlings, as well as improving root structure [33]. Additionally, applying rooting agents during the transplantation of walnuts is beneficial for increasing root length, root surface area, and survival rate of seedlings [12]. In our study, high concentrations of rooting agents significantly promoted fibrous root growth and increased LA in potted tree peonies, thereby simultaneously enhancing above- and below-ground growth of seedlings. Metarhizium anisopliae, a novel biopesticide, not only efficiently kills pests in the soil [19] but also forms a mutually beneficial symbiosis with plants [34], with the ability to promote the elongation of the roots and the formation of branches [35,36]. In this study, although Metarhizium anisopliae had the least effect on TNSCA, it could affect the formation of stems, which may be related to its ability to influence plant growth by regulating the synthesis of secondary metabolites and hormones in roots [20,37]. Therefore, the effect of Metarhizium anisopliae on root signaling in tree peonies and their growth deserves further investigation. Range analysis showed that the effects of all factors on TNSCA were in the order of root pruning > rooting agent > Metarhizium anisopliae, and the optimal combination was combination 3, which involves 25% root pruning, a rooting agent concentration of 750 mg·L⁻¹, and a Metarhizium anisopliae liquid dosage of 20 million spores·mL⁻¹. This combination produced the highest stem number, LA, biomass of leaves, old roots, fibrous roots and whole seedlings, and the greatest NSCs accumulation, thereby optimizing plant morphology and nutrient accumulation.

4.2. Characteristics of NSCs Accumulation in Potted Tree Peony

NSCs play an important role in the reconstruction of plant organs. The mode of NSCs allocation in different plants relies on their distinct survival strategies [38,39]. Compared to evergreen broadleaf trees, deciduous broadleaf trees consume significantly more NSCs for leaf and shoot growth in spring, and therefore have evolved greater NSCs storage capacity [40]. Tree peony, a deciduous shrub, sheds its shoots annually. In spring, the growth of leaves, flowers and stems requires substantial energy, which relies on well-developed storage organs to meet these demands [41]. Our results showed that during the early growth stage, the storage of NSCs in different organs of the potted tree peony was ranked from highest to lowest as follows: old roots > leaves > stems > fibrous roots. More than 60% of TNSCA accumulation was stored in old roots, and under the optimal combination (3), nearly 76% of NSCs were stored in old roots, significantly higher than in other treatments. This indicates that old roots are important storage organs for tree peonies, and suitable transplantation treatments can enhance the accumulation of NSCs in old roots.

4.3. NSCs Accumulation for Tree Peony Seedling Growth

In addition to serving as a carbon skeleton and energy source for organ reconstruction, NSCs play an important regulatory role in plant osmoregulation and stress tolerance [42], and their content is an important indicator of plant growth status and production potential [43,44]. As a perennial woody plant, tree peony exhibits a complex source–sink relationship, with transformations occurring between them throughout different seasons. During the spring germination period, leaves and flower buds serve as sink organs, and their growth depends on nutrients stored in the roots [45]. During the vigorous growth period, leaves act as source organs, producing NSCs and storing them in roots for remobilization in the following year [46,47]. Therefore, the growth of the above-ground part of the tree peony is closely connected with the amount of stored NSCs, especially in the roots. The correlation analysis revealed significant or highly significant positive correlations between morphological indicators (leaves, old roots, and fibrous roots), photosynthetic characteristics (LA, WUE, and SPAD), and NSCs accumulation in old roots and seedlings, suggesting that the growth of tree peony is strongly dependent on NSCs reserves. The results of the path analysis indicated that NSCs in the old roots directly affect the accumulation of NSCs of whole seedling, while NSCs in the fibrous roots play an indirect but important role by interacting with other indicators. Therefore, during the transplantation of tree peonies, selecting an appropriate combination (combination 3) is beneficial for promoting the rational distribution of NSCs in seedlings, thereby supporting leaf and root growth, enhancing overall NSCs accumulation, and ultimately optimizing seedling quality.

5. Conclusions

Potted cultivation is becoming an increasingly important method for engineering seedling production of tree peonies, offering a lighter, more efficient and simplified approach to cultivation practices. During the transplanting process, employing 25% root pruning combined with irrigation using a rooting agent at 750 mg·L⁻¹ and a suspension of Metarhizium anisopliae at 20 million spores·mL⁻¹ optimized plant structure, increased stem numbers, and enhanced non-structural carbohydrates (NSCs) accumulation in seedlings. These findings provide a technical foundation for producing high-quality potted tree peony seedlings. However, further investigation is required to elucidate the molecular and physiological mechanisms underlying NSCs accumulation and its interaction with mineral nutrition in potted tree peonies.