Effects of Rooting Substrates and Plant Growth Regulators on Rooting Performance, Photosynthetic Characteristics, and Soil Properties of Broussonetia × kazinoki Sieb. Cuttings

Abstract

Daknamu (Broussonetia × kazinoki), the primary fiber source for hanji (traditional Korean handmade paper), provides fibers that are highly durable and used in fine-edition publishing as well as in the conservation and restoration of cultural heritage materials and historic books. However, hanji production has declined due to decreased farm cultivation of B. × kazinoki, emphasizing the need for efficient vegetative propagation. This study evaluated the effects of three rooting media (commercial substrate, a mixture of commercial substrate and decomposed granite soil, and decomposed granite soil) and two plant growth regulators (auxins), 1-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA), including a rooting powder containing 0.8% IBA, on rooting performance and physiological responses. Decomposed granite soil produced the highest rooting rate, and the rooting effect index peaked with the rooting powder treatment. Exogenous auxins consistently increased the rooting rate and improved root traits. Photosynthetic activity was enhanced in decomposed granite soil, indicating improved water uptake following root development. Chlorophyll fluorescence showed a low F_v/F_m ratio and a JIP pattern indicative of stress. Soil analyses confirmed greater aeration and drainage in decomposed granite soil but revealed limitations in post-rooting water and nutrient availability. Root traits were positively correlated with photosynthetic parameters and available phosphorus, whereas electrical conductivity, cation-exchange capacity, moisture, organic matter, total nitrogen, and exchangeable cations were negatively correlated. Decomposed granite soil combined with 1500 mg·L⁻¹ IBA or rooting powder provided practical conditions for nursery-scale propagation. These findings provide a scientific basis for developing efficient cutting propagation systems for B. × kazinoki in farms and nurseries.

1. Introduction

Paper is one of the greatest inventions in human history [1]. In particular, the bast fibers of moraceous tree species are important raw materials for papermaking [2]. Handmade paper derived from these fibers is highly durable and is used in fine-edition publishing as well as in the conserving and restoring cultural heritage materials and historic books [3]. Hanji is a traditional Korean handmade paper with a history of more than 1500 years and is renowned for its durability and archival stability [4]. Recently, hanji has attracted global attention for its effective use in the conservation and restoration of valuable paper-based cultural heritage artifacts in Italy and the Vatican [5]. Broussonetia × kazinoki Sieb. (daknamu), is a monoecious woody plant belonging to the genus Broussonetia in the family Moraceae (Figure 1). It grows 2–5 m in height with variable leaves that maybe lobed or unlobed and usually have toothed margins, often with 2–3 deep lobed (Figure 1a). The species flowers from April to May, producing oval staminate and globose pistillate inflorescences (Figure 1b), and bears fruit in September [6]. B. × kazinoki has long been recognized as a natural hybrid between B. monoica and B. papyrifera and has been predominantly propagated vegetatively for cultivation [1,7,8]. It is distributed across Korea, Japan, and China and has been widely used for papermaking throughout East Asia [9]. Typically, one-year-old shoots of B. × kazinoki are used as raw materials for hanji production. The harvested shoots are steamed in a process known as dakmuji (Korean term) to facilitate bark removal and then dried (Figure 1c). The black bark is soaked in water to remove the outer layer, producing white bark, which is used to make hanji (Figure 1d) [5]. The long fibers of B. × kazinoki promote entanglement and increase the bonding area during papermaking, which enhances the strength of the sheet and contributes to the superior quality of hanji [10]. Moreover, B. × kazinoki exhibits various pharmacological activities, including inhibitory effects on atopic dermatitis-like responses [11] and antidiabetic and antihyperglycemic effects [12], suggesting its potential as a source of antidiabetic therapeutic [13]. However, the production base for B. × kazinoki has been shrinking in recent years due to multiple factors, including rural depopulation, aging agricultural population, rising labor costs, increasing raw material imports, and weakening demand [14]. Annual output has also become highly volatile, underscoring the urgent need to establish a stable supply system [15,16,17,18]. Therefore, systematic measures are required to secure a stable supply of high-quality B. × kazinoki and enable growers to cultivate it more efficiently.

Vegetative propagation is widely used in forestry and horticulture for clonal multiplication of elite genotypes [19]. Cutting propagation is a cost-effective technique that preserves the genetic identity of the mother plant while achieving high multiplication rates [20]. It does not require specialized facilities, allowing easy adoption by farms and nurseries [21,22]. Success in cutting depends on multiple factors, such as timing, rooting substrate, plant growth regulators (PGRs) [23], and rooting capacity; therefore, elucidating the determinants of rooting is critical [24]. In addition, the optimal propagation medium varies with species, cutting type, and season. During rooting, the propagation medium secures the cutting, supplies adequate moisture, allows air exchange, and limits excessive light transmission [25,26]. Previous studies on the mass of B. × kazinoki have reported tissue culture-based propagation systems [27], although these methods require high technical expertise under aseptic conditions. Other studies have examined the rooting characteristics of cuttings according to media composition and PGRs [28], the interaction between shading and PGRs [29], and the effects of cutting season, exogenous auxin concentration, and cutting length [30]. However, these studies varied widely in their experimental condition and ranges of PGR concentration, and few have quantitatively compared the interaction effects of substrates and growth regulators under field-relevant conditions. Moreover, the linkage between rooting indices and physiological activity has not been fully elucidated, limiting the establishment of standardized propagation protocols for practical application. Therefore, this study aimed to systematically determine the effects of substrate composition and PGR treatment on the rooting and growth traits of B. × kazinoki cuttings, providing a physiological basis for developing a stable and efficient propagation system.

2. Materials and Methods

2.1. Plant Materials

To evaluate cutting propagation of B. × kazinoki, we collected 1-year-old shoots in January 2024 from trees planted at the experimental forest of the Forest Biomaterial Research Center of National Institute of Forest Science (35°12′44″ N 128°09′26″ E). To minimize moisture loss, the shoots were wrapped in paper and vinyl, sealed, stored at 4 °C, and used in April 2024 for rooting. The experiment was conducted in a smart greenhouse at the same location. The cuttings, which were hardwood cuttings, contained four buds and were trimmed to a length 7–10 cm and a diameter of 7–9 mm. The basal end of each cutting was cut at an angle of approximately 45° (Figure 2a). Ten cuttings per treatment were planted in propagation trays (520 mm × 360 mm × 86 mm) with three replicates and arranged in a randomized block design. The trays were covered with white agricultural nonwoven fabric for 30 days to maintain heat and humidity. Watering was applied once daily using a fine mist to prevent substrate desiccation. From April to September 2024, the mean temperatures in April, May, June and July–September were 25.1 °C, 23.5 °C, 25.0 °C and above 28.0 °C, respectively. The mean relative humidity remained above 60% throughout this period (Figure 2b).

2.2. Rooting Substrate Treatments

To evaluate substrate effects on rooting, we used a fertilizer-free commercial substrate (Tosil; East Co. Ltd., Hapcheon, Republic of Korea) and decomposed granite soil (DS). The treatments consisted of three levels: commercial substrate alone (CS), decomposed granite soil alone (DS), and a 1:1 (v/v) mixture of the two (CS + DS). Each substrate was packed into a propagation tray, and pilot holes matching the cutting base diameter were created at uniform spacing before inserting the cuttings.

2.3. PGR Treatments

To assess the effects of PGRs on rooting, we used indole-3-butyric acid (IBA; Daejung C&M, Siheung, Republic of Korea) and 1-naphthaleneacetic acid (NAA; Sigma-Aldrich, St. Louis, MO, USA). Stock solutions were prepared by dissolving the acid in 1 N KOH (Daejung C&M, Siheung, Republic of Korea) and diluting with distilled water to 500, 1500 or 3000 mg∙L⁻¹. Rooting powder (RP, 0.8% IBA; Maia Products, Inc., Westlake Village, CA, USA) was applied in accordance with the manufacturer’s instructions. For the solution treatments, 30 cuttings per treatment were subjected to a 10 s quick dip; control cuttings were dipped in distilled water for 10 s. The selection of IBA and NAA concentrations (500 mg∙L⁻¹, 1500 mg∙L⁻¹ and 3000 mg∙L⁻¹), as well as the dipping method, was based on previous studies [25,31].

2.4. Leaf Gas Exchange Measurements

At 120 days after planting (DAP), leaf gas exchange was measured on five plants per treatment, with three replicates. Measurements were conducted between 10.00 and 15.00 using a portable photosynthesis system (LI-6400XT; Li-COR, Lincoln, NE, USA) on the most recently fully expanded leaf [32]. The variables were net photosynthetic rate (P_n; µmol CO₂∙m⁻²∙s⁻¹), stomatal transpiration (T_r; mmol H₂O·m⁻²·s⁻¹), and stomatal conductance (g_s; mol H₂O·m⁻²∙s⁻¹). Environmental settings were as follows: flow rate, 500 µmol·s⁻¹; reference, CO₂ 400 µmol·mol⁻¹; photosynthetic photon flux density, 1000 µmol·m⁻²·s⁻¹; leaf temperature, 25 ± 3 °C; and relative humidity, 50%–60%. Water use efficiency (WUE) was calculated as P_n/E (Equation (1)).

WUE (µmol CO₂·mmol H₂O⁻¹) = P_n/E,

(1)

2.5. JIP-Test Chlorophyll Flourescence and SPAD Mesurements

To assess photosynthetic responses to substrate and PGR treatments, JIP-test chlorophyll fluorescence was measured using a portable fluorometer (JIP chlorophyll fluorescence) on the same leaves used for gas exchange measurements between 10.00 and 15.00. Prior to fluorescence measurements, the leaves were dark-adapted for 15 min using a dark adaptation clip. Measurements of six fluorescence parameters indicative of stress were taken at 120 DAP, and their definitions and descriptions are provided in

Table 1. Definitions and equations of measured and calculated parameters derived from the chlorophyll a fluorescence transient.

Parameter	Name and Basic Physiological Interpretation
Specific energy fluxes (per active PSII reaction center)
ABS/RC = M0·(1/VJ)·(1/φP0)	Absorbed photon flux per reaction center (RC)
TR0/RC = M0·(1/VJ)	Trapped exciton flux (leading to QA reduction) of absorbed photons per RC
ET0/RC = M0·(1/VJ)·ψ0	Electron transport flux (from reduced QA to QB) per RC
DI0/RC = (ABS/RC) − (TR0/RC)	Dissipated energy flux per RC
Performance index
PIABS = (RC/ABS)·[φP0/(1 − φP0)]·[ψE0/(1 − ψE0)]	Performance index for energy conservation from photons absorbed by PSII antenna, to the reduction of QB
Quantum yields and probabilities
φP0 = Fv/Fm	Maximum quantum efficiency of primary PSII photochemistry; maximum efficiency at which light absorbed by PSII is used for reduction of QA
ψE0 = 1 − VJ	Probability with which a PSII trapped election is transferred from reduced QA to QB

[33,34].

Chlorophyll content was determined non-destructively using SPAD-502plus meter (Konica Minolta, Tokyo, Japan). Measurements were made on the same leaves at 90, 120 and 150 days after cutting, and the mean of three positions per leaf was used for analysis.

2.6. Root Growth Measurement

Root growth was evaluated at 180 DAP. The measured variables were rooting rate, rooting effect index (REI), root number (per cutting), mean and maximum root diameter, and mean and maximum root length. Root diameter was measured using a Vernier caliper (CD-15APX; Mitutoyo Corp., Kawasaki, Japan). The rooting rate and REI were calculated as described by Sun et al. [35] (Equations (2) and (3)).

Rooting rate (%) = (Number of rooted cuttings/Total number of cuttings) × 100

(2)

REI = (Average root length × Number of rooted cuttings)/Total number of cuttings

(3)

2.7. Analysis of Rooting Substrates

The physical properties of the substrates were analyzed before and after cultivation using samples air-dried at room temperature and sieved to 2 mm [36]. Bulk density and moisture content were determined by oven drying at 105 °C and calculating mass difference. The chemical properties of the samples were also measured. pH and electrical conductivity (EC) were determined using a 1:5 soil:distilled water suspension. Cation exchange capacity (CEC) and exchangeable cations (K⁺, Ca²⁺, Mg²⁺, and Na⁺) were extracted with 1 N NH₄OAc, and the exchangeable cations were quantified by ICP-OES (SPECTROBLUE, SPECTRO Analytical Instruments, Kleve, Germany). Organic matter (OM) content was determined using the Walkley–Black method [37], total nitrogen (TN) content using the Kjeldahl method [38], and available phosphorus (Av.P₂O₅) content using the Lancaster method [39]. The pre-cultivation chemical properties of the substrates are summarized in

Table 2. pH, electrical conductance (EC), cation exchange capacity (CEC) bulk density, moisture, organic matter (OM), total nitrogen (TN), available phosphorus (Av.P₂O₅), and exchangeable cations (Exch. cation) of the different media before planting.

Soil		CS	CS + DS	DS
pH [1:5]	v/v	7.34	7.25	7.47
EC	dS·m−1	0.47	0.26	0.06
CEC	cmol+·kg−1	28.20	6.08	2.26
Bulk density	g·mL−1	0.31	0.83	1.28
Moisture	%	61.04	33.19	5.90
OM		16.09	3.75	0.80
Total N		0.14	0.04	0.01
Av.P2O5	mg·kg−1	7.21	6.01	7.67
K+	cmol+·kg−1	2.19	0.24	0.05
Ca2+		8.05	2.01	2.10
Mg2+		6.03	0.98	0.19
Na+		8.37	1.13	0.11

Note: CS, commercial substrate; CS + DS, commercial substrate+ decomposed granite soil; and DS, decomposed granite soil.

.

2.8. Statistical Analysis

All statistical analyses were performed using IBM SPSS Statistics (version 29.0; IBM Corp., Armonk, NY, USA). Two-way analysis of variance (ANOVA) was used to examine the main and interaction effects of the rooting substrate (S) and plant growth regulator (P). When significant effects were detected, mean separation were perfomed using Duncan’s multiple range test at p < 0.05. Linear associations among variables were evaluated using Pearson’s correlation analysis.

3. Results

3.1. Leaf Gas Exchange

Significant difference in the photosynthetic parameters of the cutting leaves were observed among substrate types (two-way ANOVA, factor S: P_n, F = 14.62 ***; g_s, F = 7.98 ***; T_r, F = 7.84 ***; WUE, ns) (

Table 3. Photosynthetic parameters of B. × kazinoki according to substrate type and plant growth regulator concentrations at 120 days after planting (n = 3).

Substrate Type	Plant Growth Regulator (mg·L−1)	Pn	gs	Ci	Tr	WUE
		(µmol CO2·m−2·s−1)	(mol m−2·s−1)	(µmol CO2·mol−1)	(mmol·m−2·s−1)	(µmolCO2·mmol H2O−1)
CS	0	0.77 c,d,e	0.017 a,b	381.04	0.55 a,b	2.22
	NAA500	1.27 b,c,d,e	0.013 b	270.09	0.76 a,b	3.65
	NAA1500	0.22 e	0.000 b	238.45	0.06 a,b	2.86
	NAA3000	0.38 d,e	0.000 b	533.93	0.09 a,b	1.97
	IBA500	0.36 d,e	0.003 b	302.72	0.14 a,b	2.77
	IBA1500	1.14 b,c,d,e	0.017 a,b	350.75	0.64 a,b	1.97
	IBA3000	0.96 b,c,d,e	0.007 b	399.21	0.31 a,b	2.21
	RP	0.29 d,e	0.003 b	177.56	0.23 a,b	2.54
CS + DS	0	0.31 d,e	0.000 b	207.00	0.09 a,b	1.26
	NAA500	1.23 b,c,d,e	0.007 b	89.63	0.15 a,b	7.25
	NAA1500	3.21 a,b,c,d	0.027 a,b	353.97	1.83 a,b	1.26
	NAA3000	0.63 c,d,e	0.010 b	193.65	0.32 a,b	1.24
	IBA500	2.19 a,b,c,d,e	0.027 a,b	380.43	1.56 a,b	0.91
	IBA1500	0.93 b,c,d,e	0.017 a,b	457.93	0.85 a,b	1.20
	IBA3000	0.71 c,d,e	0.003 b	392.48	0.09 a,b	2.17
	RP	0.32 d,e	0.003 b	98.60	0.03 b	0.73
DS	0	2.05 a,b,c,d,e	0.073 a	256.73	1.63 a,b	3.25
	NAA500	2.31 a,b,c,d,e	0.037 a,b	310.32	1.07 a,b	1.99
	NAA1500	2.73 a,b,c,d,e	0.027 a,b	202.02	1.32 a,b	1.76
	NAA3000	3.53 a,b,c	0.006 b	349.89	2.04 a	1.75
	IBA500	2.19 a,b,c,d,e	0.023 a,b	196.93	0.68 a,b	3.96
	IBA1500	4.54 a	0.030 a,b	186.88	2.03 a,b	2.76
	IBA3000	1.93 a,b,c,d,e	0.020 a,b	315.73	1.10 a,b	4.94
	RP	3.83 a,b	0.047 a,b	232.95	1.68 a,b	3.57
Substrate type (S)		14.62 ***	7.98 ***	ns	7.84 ***	7.84 ***
Plant growth regulator (P)		ns	ns	ns	ns	ns
(S) × (P)		ns	ns	ns	ns	ns

Note: CS, commercial substrate; CS + DS, commercial substrate mixed with decomposed granite soil; DS, decomposed granite soil; P_n, net photosynthetic rate; g_s, stomatal conductance; C_i, intercellular CO₂ concentration; T_r, transpiration rate; WUE, water-use efficiency. Means within a column followed by the same letter are not significantly different at the 5% level according to Duncan’s multiple range test (p < 0.05). ns, and *** indicate non-significant and significant differences at p < 0.001, respectively.

). By contrast, neither the main effects of P nor the S × P interaction was significant for any photosynthetic variable. Among substrates, P_n, g_s and T_r were significantly higher in DS and CS + DS than in CS alone, whereas WUE showed no significant difference. The ranking of P_n followed the order DS > CS + DS > CS, with mean values ranging from 1.93–4.54 µmol CO₂·m⁻²·s⁻¹ in DS, 0.31–3.21 µmol CO₂·m⁻²·s⁻¹ in CS + DS, and 0.22–1.27 µmol CO₂·m⁻²·s⁻¹ in CS. The highest P_n was observed in DS treated with IBA 1500 mg·L⁻¹ (4.54 µmol CO₂·m⁻²·s⁻¹). Similarly, g_s was greatest in DS without PGR application (0.073 mol·m⁻²∙s⁻¹), and T_r tended to be higher in DS treatments (0.68–2.04 mmol m⁻²·s⁻¹). These results suggest that the DS promoted gas exchange activity compared with the CS, regardless of PGR treatment.

3.2. SPAD and JIP Analyses of B. × kazinoki Cuttings

3.2.1. SPAD

Leaf SPAD values differed significantly among substrate types at 90, 120 and 150 DAP (p < 0.001, Figure 3). At 90 DAP, SPAD was higher in CS (31.90–38.64) than in DS (28.08–35.19), with CS + DS showing intermediate values. SPAD declined over time across all substrates; the decline was particularly pronounced in DS, which reached 16.74–26.23 by 150 DAP. Neither the main effect of the PGR nor the S × PGR interaction was significant at any time point, indicating that under our conditions, PGR treatment did not directly affect chlorophyll status.

3.2.2. JIP-Test Analysis

Across treatments, F_v/F_m was generally low (0.52–0.65) and did not differ (

Table 4. Photosynthetic response of B. × kazinoki according substrate type and plant growth regulator concentrations at 120 days after planting (n = 3).

Substrate Type	Plant Growth Regulator (mg·L−1)	Fv/Fm	PIABS	ABS/RC	TR0/RC	ET0/RC	DI0/RC
CS	0	0.66	0.53 a	2.60 c,d	1.70 b,c,d	0.66	0.90 b
	NAA500	0.60	0.38 a,b,c	3.16 a,b,c,d	1.80 a,b,c	0.63	1.36 a,b
	NAA1500	0.58	0.34 a,b,c	3.14 a,b,c,d	1.74 b,c,d	0.64	1.40 a,b
	NAA3000	0.60	0.39 a,b,c	3.04 a,b,c,d	1.77 b,c,d	0.62	1.27 a,b
	IBA500	0.65	0.47 a,b	2.74 b,c,d	1.75 b,c,d	0.64	0.99 b
	IBA1500	0.62	0.39 a,b,c	2.93 a,b,c,d	1.80 a,b,c	0.64	1.13 a,b
	IBA3000	0.63	0.50 a	3.09 a,b,c,d	1.90 a,b,c	0.69	1.19 a,b
	RP	0.60	0.41 a,b,c	2.55 c,d	1.64 c,d	0.59	0.91 b
CS + DS	0	0.52	0.33 a,b,c	2.37 d	1.46 d	0.56	0.90 b
	NAA500	0.66	0.50 a	2.47 c,d	1.63 c,d	0.62	0.84 b
	NAA1500	0.61	0.32 a,b,c	3.02 a,b,c,d	1.82 a,b,c	0.59	1.19 a,b
	NAA3000	0.63	0.43 a,b,c	2.77 b,c,d	1.73 b,c,d	0.64	1.05 a,b
	IBA500	0.66	0.55 a	2.55 c,d	1.67 c,d	0.68	0.88 b
	IBA1500	0.64	0.46 a,b	2.74 b,c,d	1.71 b,c,d	0.66	1.03 a,b
	IBA3000	0.64	0.45 a,b	2.87 a,b,c,d	1.80 a,b,c	0.67	1.07 a,b
	RP	0.62	0.37 a,b,c	3.08 cd	1.84 a,b,c	0.65	1.23 a,b
DS	0	0.60	0.32 a,b,c	3.27 a,b,c	1.90 a,b,c	0.63	1.37 a,b
	NAA500	0.65	0.39 a,b,c	2.83 a,b,c,d	1.82 a,b,c	0.62	1.01 b
	NAA1500	0.57	0.21 b,c	3.68 a	2.01 a,b	0.50	1.67 a
	NAA3000	0.62	0.32 a,b,c	3.12 a,b,c,d	1.89 a,b,c	0.65	1.23 a,b
	IBA500	0.65	0.40 a,b,c	2.78 b,c,d	1.80 a,b,c	0.64	0.98 b
	IBA1500	0.59	0.18 c	3.59 a,b	2.11 a	0.60	1.49 a,b
	IBA3000	0.61	0.41 a,b,c	3.12 a,b,c,d	1.88 a,b,c	0.68	1.24 a,b
	RP	0.62	0.28 a,b,c	3.08 a,b,c,d	1.89 a,b,c	0.59	1.19 a,b
Substrate type (S)		ns	5.63 **	6.26 **	10.24 ***	ns	3.41 *
Plant growth regulator (P)		ns	ns	ns	ns	ns	ns
(S) × (P)		ns	ns	ns	ns	ns	ns

Note: CS, commercial substrate; CS + DS, commercial substrate mixed with decomposed granite soil; DS, decomposed granite soil. Means within a column followed by the same letter are not significantly different at the 5% level according to Duncan’s multiple range test (p < 0.05). ns, *, **, and *** indicate non-significant or significant differences at p < 0.05, 0.01, and 0.001, respectively.

). By contrast, the main effect of the substrate was significant for PI_ABS (F = 5.63, p < 0.01), ABS/RC (F = 6.26, p < 0.01), TR₀/RC (F = 10.24, p < 0.001), and DI₀/RC (F = 3.41, p < 0.05), indicating that the substrate environment influenced PSII-related fluorescence indices. Pairwise comparisons showed that compared with CS, DS exhibited higher ABS/RC (+9.5%), TR₀/RC (+8.5%), and DI₀/RC (+11.3%) and lower PI_ABS (−26.4%, range 0.18–0.41). Compared with CS + DS, DS also showed higher ABS/RC (+16.5%), TR₀/RC (+12.0%), and DI₀/RC (+24.3%).

3.3. Rooting Characteristics of B. × kazinoki

The morphological characteristics of roots are shown in Figure 4. Rooting rate differed markedly among the substrates (Figure 5a). The rooting rete in DS averaged 30.18 ± 2.37%, which was 3.4-fold higher than that in CS (8.95 ± 2.37%) and 6.0-fold higher than that in CS + DS (5.04 ± 2.37%) (F = 32.69, p < 0.001). In multiple comparisons, DS IBA1500 showed the highest rooting rate of 46.67%, which was approximately 7.0-fold higher than that in CS without PGR and 1.5-fold higher than that in DS without PGR. In CS, the RP treatment yielded approximately 23.3%, which was approximately 3.5-fold higher than that in CS without PGR. In CS + DS, no rooting was observed in the absence of PGR, NAA500, and RP. Moreover, NAA and IBA at 3000 mg∙L⁻¹ reduced rooting, whereas those at 500 mg∙L⁻¹ did not differ from that without PGR, suggesting a unimodal (bell-shaped) PGR dose response within the test range.

Similarly, the REI was highest in DS (Figure 5b). Two-way ANOVA showed significant effects of the substrate (F = 42.52, p < 0.001), PGR (F = 6.69, p < 0.001), and their interaction (F = 4.92, p < 0.001). Based on estimated means, the REI in DS was 2.36 ± 0.19, which was approximately 7.6-fold higher than that in CS (0.31 ± 0.19) and 15.7-fold higher than that in CS + DS (0.15 ± 0.19). Within DS, RP achieved the highest REI of 6.42 ± 0.53, which was approximately 26.4-fold greater than that in DS without PGR, followed by DS IBA 1500 (4.18 ± 0.53) and DS IBA 3000 (3.39 ± 0.53). These results indicate that the physicochemical properties of the substrate are decisive for rooting and that PGR efficacy is strongly modulated by substrate conditions. Therefore, securing optimal substrate conditions is a prerequisite for the reliable PGR-mediated promotion of rooting.

The number of roots per cutting was highest in DS RP (5.56), followed by DS IBA 1500 (3.07), DS NAA 3000 (1.90), DS NAA 1500 (1.70), and DS IBA 3000 (1.53) (Figure 6a). The mean root diameter was 1.26 mm in DS RP, which was approximately 7.8-fold greater than that in DS without PGR (Figure 6b). In CS, CS IBA 3000 showed the highest mean root diameter of 0.68 mm, which was approximately 9-fold higher than that in CS without PGR. The maximum root diameter was also greatest in DS RP (1.97 mm) (Figure 6c). The mean root length in DS RP reached 16.86 cm, which was 15.9-fold higher than that in DS without PGR. The subsequent ranks were as follows: DS IBA 3000 (10.78 cm) and DS IBA1500 (8.30 cm) (Figure 6d). Similarly, the maximum root length was greatest in DS RP (34.49 cm) (Figure 6e). Notably, in CS + DS, the range of maximum root length was only 0–0.15 cm, indicating very poor elongation.

3.4. Physicochemical Analysis of Rooting Substrates

The substrates used in this study differed markedly in both nutrient composition (N, P and K) and physical properties (

Table 5. Soil characteristics under substrates types and plant growth regulator (PGR) concentrations after 180 days of B. × kazinoki planting (n = 3).

Substrate Type	Plant Growth Regulator (mg·L−1)	pH	EC	CEC	Bulk Density	Moisture	OM	TN	Av.P2O5	K+	Ca2+	Mg2+	Na+
			(dS·m−1)	(cmol+·kg−1)	(g·mL−1)	(%)			(mg·kg−1)	(cmol+·kg−1)
CS	0	7.40 b,c,d,e,f	0.21 a,b,c,d,e	18.83 a,b	0.48 h	76.65 a,b	9.27 a	0.08 c	5.18 c	1.34 a,b,c	6.16 a,b	3.93 a	3.88 b,c
	NAA500	7.41 b,c,d,e,f	0.21 a,b,c,d	18.55 b	0.49 h	78.91 a	9.25 a	0.09 b,c	5.25 c	1.07 c	5.67 b	3.91 a	2.90 d
	NAA1500	7.48 a,b,c	0.23 a,b	19.60 a,b	0.48 h	75.21 b	9.19 a	0.11 a	4.88 c	1.52 a	5.82 a,b	3.92 a	4.87 a
	NAA3000	7.50 a,b	0.23 a	20.19 a	0.51 h	76.59 a,b	9.17 a	0.09 c	5.03 c	1.44 a,b	5.99 a,b	3.85 a	4.39 a,b
	IBA500	7.56 a	0.23 a,b	19.39 a,b	0.50 h	77.85 a,b	9.33 a	0.09 b,c	4.99 c	1.45 a,b	5.89 a,b	3.93 a	4.21 a,b
	IBA1500	7.46 a,b,c,d,e	0.23 a,b	20.28 a	0.49 h	76.94 a,b	9.44 a	0.10 a,b	5.10 c	1.50 a	6.19 a,b	3.87 a	4.35 a,b
	IBA3000	7.44 a,b,c,d,e	0.22 a,b,c	19.05 a,b	0.50 h	76.22 a,b	9.24 a	0.09 b,c	5.07 c	1.19 b,c	6.39 a	3.90 a	3.45 c,d
	RP	7.37 b,c,d,e,f,g,h	0.23 a,b	19.32 a,b	0.48 h	77.01 a,b	8.46 b	0.09 c	5.28 c	1.45 a,b	6.24 a,b	3.92 a	4.18 a,b
CS + DS	0	7.34 e,f,g,h	0.18 d,e,f,g,h	9.87 c	0.81 g	32.61 c	3.93 c	0.03 d	6.16 b	0.39 d	4.34 c	1.40 c	1.64 e
	NAA500	7.39 b,c,d,e,f,g	0.19 c,d,e,f,g,h	7.93 d,e	0.84 e,f	30.46 c,d,e	3.39 d,e	0.03 d	6.47 b	0.34 d,e	4.12 c,d	1.67 b	1.68 e
	NAA1500	7.39 b,c,d,e,f,g	0.19 c,d,e,f,g,h	7.72 e	0.82 e,f,g	30.93 c,d	3.46 d	0.03 d	6.39 b	0.33 d,e	4.02 c,d	1.42 c	1.67 e
	NAA3000	7.36 c,d,e,f,g,h	0.19 a,b,c,d,e,f,g	7.99 d,e	0.83 e,f,g	27.76 e,f	3.21 d,e	0.03 d	6.14 b	0.45 d	4.08 c,d	1.43 c	1.86 e
	IBA500	7.40 b,c,d,e,f	0.20 a,b,c,d,e,f	8.81 c,d,e	0.85 e	29.27 d,e	3.56 c,d	0.03 d	6.60 b	0.27 d,e	3.60 d,e	1.27 c	1.28 e
	IBA1500	7.48 a,b,c,d	0.19 b,c,d,e,f,g	9.75 c	0.82 f,g	29.66 c,d,e	3.96 c	0.04 d	6.09 b	0.43 d	3.67 d,e	1.42 c	1.87 e
	IBA3000	7.31 f,g,h	0.19 c,d,e,f,g,h	9.31 c,d	0.85 e	30.45 c,d,e	3.98 c	0.04 d	6.18 b	0.31 d,e	3.37 e	1.30 c	1.48 e
	RP	7.40 b,c,d,e,f	0.17 d,e,f,g,h	8.18 d,e	0.84 e,f	25.37 f	3.00 e	0.03 d	6.63 b	0.32 d,e	3.70 d,e	1.32 c	1.46 e
DS	0	7.39 b,c,d,e,f	0.16 f,g,h	2.187 f	1.25 a,b	7.11 g	0.65 f	0.01 e	7.93 a	0.03 e	1.98 e	0.15 d	0.22 f
	NAA500	7.42 b,c,d,e,f	0.16 f,g,h	2.024 f	1.24 a,b	6.82 g	0.70 f	0.01 e	7.56 a	0.03 e	2.15 e	0.16 d	0.24 f
	NAA1500	7.41 b,c,d,e,f	0.17 e,f,g,h	2.14 f	1.25 a	6.05 g	0.68 f	0.01 e	7.50 a	0.02 e	1.99 e	0.15 d	0.23 f
	NAA3000	7.35 d,e,f,g,h	0.16 f,g,h	1.98 f	1.22 b,c	6.96 g	0.72 f	0.01 e	7.93 a	0.02 e	2.16 e	0.15 d	0.27 f
	IBA500	7.30 f,g,h	0.17 d,e,f,g,h	2.06 f	1.24 a,b	6.21 g	0.74 f	0.01 e	7.88 a	0.02 e	2.13 e	0.16 d	0.23 f
	IBA1500	7.26 h	0.15 gh	2.36 f	1.21 c	7.08 g	0.77 f	0.01 e	7.78 a	0.02 e	2.01 e	0.14 d	0.22 f
	IBA3000	7.26 g,h	0.15 h	2.04 f	1.20 c	6.61 g	0.76 f	0.01 e	8.12 a	0.03 e	2.19 e	0.15 d	0.24 f
	RP	7.45 a,b,c,d,e	0.16 f,g,h	2.29 f	1.15 d	6.75 g	0.74 f	0.01 e	7.55 a	0.03 e	2.11 e	0.18 d	0.26 f
Substrate type (S)		14.23 ***	53.51 ***	2564.06 ***	13,842.11 ***	10,764.60 ***	7152.29 ***	580.18 ***	249.35 ***	434.04 ***	949.18 ***	6670.14 ***	500.68 ***
Plant growth regulator (P)		ns	ns	ns	7.10 ***	ns	6.19 ***	ns	ns	ns	ns	ns	2.56 *
(S) × (P)		3.19 **	ns	ns	6.69 ***	2.22 **	2.36 *	ns	ns	ns	2.27 *	ns	2.25 *

Note: CS, commercial substrate; CS + DS, commercial substrate mixed with decomposed granite soil; DS, decomposed granite soil. Means within a column followed by the same letter are not significantly different at the 5% level according to Duncan’s multiple range test (p < 0.05). ns, *, **, and *** indicate non-significant or significant differences at p < 0.05, 0.01, and 0.001, respectively.

). The substrate type significantly affected all measured properties, indicating that substrate selection influenced the physicochemical environment relevant to rooting. PGR application also had significant effects on bulk density, OM, and Na⁺ contents (p < 0.05). Among the physical traits, bulk density was highest in DS (1.20 g∙mL⁻¹), intermediate in CS + DS (0.83 g∙mL⁻¹), and lowest in the CS (0.50 g∙mL⁻¹), whereas moisture content showed the opposite pattern, with 76.93% in CS and 6.70% in DS. Similarly, CEC and OM were greatest in CS and lowest in DS, with CS + DS showing intermediate values. The TN content in CS (0.08–0.11%) was approximately 8-fold higher than that in DS (0.01%) (F = 580.18, p < 0.001). By contrast, Av.P₂O₅ was greatest in DS (7.50–8.12 mg∙kg⁻¹), followed by CS + DS (6.09–6.63 mg∙kg⁻¹) and CS (4.88–5.28 mg∙kg⁻¹) (F = 249.35, p < 0.001). Overall, the physicochemical properties of CS and DS exhibited contrasting trends, while those of CS + DS were intermediate between the two.

3.5. Correlations Among Gas Exchanges, Chlorophyll Fluorescence, Soil Factors, and Rooting Traits

We conducted correlation analyses to evaluate the relationships among rooting rate, REI, root growth traits, leaf gas exchange (P_n, g_s, C_i, T_r and WUE), JIP-test fluorescence indices, SPAD at multiple time points, soil properties and PGR treatments (Figure 7). Root growth traits positively correlated with P_n, g_s, and T_r. Notably, P_n vs. rooting rate (r = 0.55, p < 0.01) and T_r vs. rooting rate (r = 0.53, p < 0.01) were significantly correlated, indicating enhanced photosynthetic performance with root development. In contrast, at 90, 120 and 150 DAP, SPAD values showed significant negative correlations with rooting-related traits, particularly at 150 DAP where SPAD was correlated with rooting rate (r = −0.66, p < 0.01). These results indicate that higher chlorophyll content during the later rooting phase was associated with lower root formation efficiency and reduce elongation.

Among the JIP indices, ABS/RC (r = 0.45, p < 0.01), TR₀/RC (r = 0.53, p < 0.01), and DI₀/RC (r = 0.36, p < 0.01) were positively correlated with the rooting rate, whereas PI_ABS (r = −0.408, p < 0.01) and ET₀/RC (r = −0.308, p < 0.01) were negatively correlated. Furthermore, EC, CEC, moisture content, OM, TN, and exchangeable cations exhibited negative correlations with rooting traits, suggesting that water-and nutrient-retentive substrates inhibit rooting, while well-drained, low-nutrient media facilitate root induction.

Among the PGR treatments, RP exhibited significant positive correlations with REI (r = 0.35, p < 0.01), root number per plant (r = 0.37, p < 0.01), root diameter and length, confirming its effectiveness in promoting root initiation and elongation. Conversely, NAA and IBA treatments showed weak and non-significant correlations with most rooting trait, suggesting limited contribution of exogenous auxin concentration to rooting under the tested substrate condition.

4. Discussion

Clear differences among the rooting substrates were evident, with DS generally enhancing P_n, g_s, and T_r. This finding agrees with previous reports that Cordia alliodora cutting can photosynthesize even before rooting (0.47–4.96 µmol CO₂∙m⁻²∙s⁻¹) [40] and that photosynthesis and g_s increase sharply as roots elongate after rooting in poinsettia (Euphorbia pulcherrima) and red maple (Acer rubrum) [41,42]. Consistently, we observed positive correlations between rooting and gas exchange variables. Thus, new root formation restores water supply, which can activate photosynthesis. During the cutting propagation process, desiccation of cutting tissue before root initiation can easily induce water stress and stomatal closure [43,44]. Carbohydrates produced by photosynthesis supply both energy and structural resources to the root initials, promoting rooting [43]. Thus, adventitious root formation involves a high demand for carbohydrate [45], with the required energy derived both from store reserves and from the photosynthetic assimilates produced by cutting [46,47]. Backhousia citriodora cuttings with mature leaves root faster, implying dependence on photosynthetic status [48]. In Broussonetia papyrifera, leaf retention does not alter rooting rate but affects root system growth [49]. Overall, leaf gas exchange is tightly coupled with rooting leaf management to mitigate water stress in the early stages, whereas the recovery of photosynthesis and carbohydrate supply becomes key as rooting progresses.

SPAD is closely related to the total chlorophyll content [50] and is typically lower in newly expanded leaves than in mature leaves [51]. In the present study, we measured SPAD on the uppermost fully expanded leaf at each time point. Given the higher rooting and vigorous sprouting in DS, the presence of younger leaves may partly explain the lower SPAD in DS. Additionally, the low TN content and water retention in DS may have limited chlorophyll synthesis [52]. However, Zakaria et al. [53] reported that root restriction in Capsicum annuum reduced photosynthetic rate and total leaf area, while the relative SPAD value temporarily increased under restricted growth condition. The increase in SPAD was attributed to smaller and thicker leaves, which led to a higher chlorophyll density without a direct association with photosynthetic efficiency or rooting activity. In addition, root restriction inhibited assimilates transport, resulting in the accumulation of carbohydrates in the stem instead of sufficient translocation to the roots. Therefore, SPAD can serve as a useful auxiliary indicator reflecting the nutritional status during the early rooting stage of B. × kazinoki, but it is limited in predicting rooting success on its own. The temporal decrease in SPAD value can be interpreted as a result influence by shooting activity, rooting status, nitrogen accumulation, and the assimilate partitioning structure, in conjunction with the nutritional properties of the substrate.

In the present study, the F_v/F_m values were below 0.7, lower than the typical 0.78–0.84 for healthy leaves [54,55], indicating insufficient rooting and/or water stress [56]. By contrast, PI_ABS, ABS/RC, TR₀/RC, and DI₀/RC differed significantly among substrates, whereas the effects of PGR and PGR × substrate interaction were not significant, highlighting the predominant role of the substrate environment in determining PSII performance. PI_ABS integrates RC density, primary photochemistry, and electron transport [57,58], and is more responsive than F_v/F_m under stress [34,59]. ABS/RC, TR₀/RC, and DI₀/RC typically increase under stress, whereas ET₀/RC decreases [60,61]. Performance indices such as PI_ABS or P_total can help distinguish rooting efficiency from nutritional status; however, single fluorescence parameters rarely explain rooting success [62]. Notably, DS IBA1500 exhibited high rooting but low PI_ABS, in contrast to Hedera algeriensis, in which high PI_ABS was associated with vigorous rooting [56]. These discrepancies likely stem from substrate properties; DS increased the rooting rate but was accompanied by generally lower photosynthetic efficiency, indicating physiological stress. Therefore, during establishment, proper substrate management and supportive measures are required to mitigate stress.

Correlation analyses showed that ABS/RC (r = 0.45, p < 0.01), TR₀/RC (r = 0.53, p < 0.01), and DI₀/RC (r = 0.36, p < 0.01) positively correlated with rooting rate, whereas PI_ABS (r = −0.41, p < 0.01) and ET₀/RC (r = −0.31, p < 0.01) negatively correlated with rooting rate. This finding aligns with prior research linking lower ET₀/RC to higher rooting efficiency [62] and indicates that PSII electron transport efficiency does not necessarily scale with rooting success. In other words, increases in RC-level absorption and trapping fluxes (ABS/RC, TR₀/RC) and dissipation (DI₀/RC) reflect the physiological adjustment and reallocation of the excitation energy accompanying rooting, whereas ET₀/RC may not increase concomitantly. Given the low PI_ABS, improved water and N management may help restore chlorophyll vitality.

Although the PGRs did not exert significant effects on rooting rate, they improved root morphology such as length, diameter, and number. Reports indicating that sandy substrates enhance rooting in B. kazinoki [28] are consistent with our finding that DS without PGR outperformed CS without PGR by 216%. The overall rooting rate was low, similar to the 33.3% reported for hardwood cuttings [30]. However, DS IBA1500 treatment achieved a 1.4-fold higher rooting rate than that observed in previous research, underscoring its practical value. The inherently low rooting tendency of B. × kazinoki likely reflects a callus-mediated rooting pathway suggested by Liu et al. [63], emphasizing the need to systematically optimize propagation conditions. According to Husen et al. [31], 3000 mg∙L⁻¹ IBA was identified as the optimal concentration for inducing adventitious roots in Morus alba cuttings. Exogenous auxin application increased the auxin concentration in the rooting zone and promoted enzyme activities associated with root initiation. Furthermore, PGRs stimulate the initial meristem activities and also motivate the growth and development of the formed roots [25,64]. During the rooting process, change in endogenous hormone balance occur, therefore exogenous hormone application is known to be one of the most effective methods for promoting adventitious root formation [65]. Among exogenous auxins, IBA is reported to be most effective [66]. In Castanea sativa, IBA treatment increased the endogenous IAA content, suggesting that IBA promotes adventitious root development by modulating endogenous auxin [67]. Hence, the appropriate application of exogenous hormones effectively promotes rooting in various woody species by triggering root-induction signals and acting as growth regulators during the expression phase [68]. In stem cutting propagation, the dipping method is the most common approach for including root adventitious formation, in which cuttings are briefly immersed in a high concentration auxin solution. The auxin molecules penetrate the plant tissues through the cut surface and are subsequently taken up by cell via pH trapping or auxin influx carrier activity [66,67]. In our concentration-response study, IBA and NAA (0–3000 mg∙L⁻¹) showed unimodal trends near 1500 mg∙L⁻¹. RP improved rooting and root traits compared with the control in DS and CS. However, excessive auxin can induce ethylene overproduction and inhibit growth; therefore, determining the optimal concentration is critical [69,70].

Substrate type emerged as the primary determinant of rooting, consistent with the notion that the physicochemical properties of the medium influence root development [71]. Roots continuously explore new soil spaces, and genes that promote root hair development are activated under low nutrient conditions [72]. Strawberry cuttings benefit from granite substrate [73], and fig [74] also respond positively to sandy mixes.

DS features irregular particles, low water holding capacity, and high macroporosity, favoring drainage and aeration but risking desiccation. CS is OM-rich, with high fine porosity, large surface area, and low bulk density [75]. CS + DS balanced these traits. While high CEC in CS enhances nutrient retention, excess water retention can limit O₂ supply and accelerate the consumption of stored carbohydrates, hindering root growth [76,77]. Elevated Na⁺ levels in CS may also reduce aeration and permeability, negatively affecting root and seedling growth [78]. Hence, adequate aeration and drainage are key for cutting propagation [79]. In hemp, porosity and bulk density are decisive, with a preference for well-aerated, OM-rich substrates [26].

Chemically, CS decreases from pre- to post-cultivation possibly because the clay-rich, high-plasticity substrate shrinks upon drying and produces more leachate [80,81]. The propagation medium should be firm/dense enough to hold cuttings, show minimal volumetric changes, and typically have a C:N ratio of 20:1 [25]. CS + DS exhibited intermediate properties, implying that the mix ratio can modulate the growth and nutritional responses. Although a perlite:peat:coconut (1:1:1, v/v/v) mixture has been reported to be optimal for B. papyrifera [82], our mixed medium produced poor rooting. Poinsettia and chrysanthemum root well under adequate N, whereas geranium depends more on carbohydrates and may suffer rooting inhibition at high N concentrations [48,83]. This can be interpreted as an effect of increased nitrogen supply reducing carbohydrate content in the cuttings and increasing carbon consuming during assimilation, which acted disadvantageously for rooting [83,84]. B. × kazinoki is likely to possess relatively higher tissue carbohydrate concentrations due to its strong sprouting ability [46], suggesting that it is more carbohydrate-dependent than nitrogen-dependent. However, quantitative evidence supporting this hypothesis remains limited, and time-series measurements of soluble sugar and starch contents, as well as the developmental stages of rooting are required to clarify these relationships. Factorial follow-up experiments varying cutting age, leaf retention, and shading are warranted. Overall, DS substrates are appear more suitable than CS or their combination for B. × kazinoki, and treatment with IBA at 1500 mg∙L⁻¹ or RP are expected to promote rooting and enhance root quality.

5. Conclusions

This study analyzed the physiological indicators related to rooting to improve the cutting propagation efficiency of B. × kazinoki and to support a stable supply system. Among the substrates in this study, DS consistently increased P_n, g_s, and T_r during the rooting period. JIP-test fluorescence indicated an overall stress status during establishment, underscoring the need for substrate management and supportive measures to alleviate stress during the early rooting stage. SPAD values declined over time of the rooting and were particularly low in DS, a pattern better explained by shoot emergence, rooting status, and soil nutrient-moisture characteristics than by time alone. Also, applying appropriate PGR concentrations to an optimal substrate can enhance root growth and quality. Therefore, the success of the entire rooting process depends on the optimal physiological condition of each rooting phase. We recommend using a well-drained, well-aerated DS combined in combination with 1500 mg∙L⁻¹ IBA or RP, along with careful early stage management, to prevent over- and under-watering and maintain chlorophyll vitality. Collectively, these findings provide a scientific foundation for developing a reliable, large-scale propagation protocol for B. × kazinoki.