Effect of Phytoregulatory Substances on Adventitious Rooting of Grapevine Rootstock Paulsen 1103 Cuttings Under Hydroponic Conditions

Abstract

In the present study, the propagation ability of rootstock 1103 Paulsen in a hydroponic system was investigated. In the first part of the experiment, the effects of indolebutyric acid (IBA), dopamine (L-DOPA), and their combination on rhizogenesis were examined. The experiment was conducted under controlled conditions in a hydroponic system. Key parameters evaluated included rooting percentage, average root diameter, average number of roots per cutting, total root area, total root length, and moisture content of the cuttings. Results showed that L-DOPA treatment, followed by the IBA + L-DOPA combination, exhibited the most favorable outcomes across these parameters. The hydroponic system proved highly effective for root formation compared to other substrates (e.g., peat, perlite, sand, or their combinations), provided that continuous aeration of the water was ensured for adequate oxygenation. The second part of the experiment focused on the response of phenolic compounds, antioxidants, sugars, and starch in woody cuttings subjected to different treatments (control, IBA, L-DOPA, and IBA + L-DOPA) and how these compounds varied over time. The objective was to assess the influence of the treatments on both the rhizogenesis process and the biochemical profile of the cuttings throughout the experiment. This research aims to contribute to the understanding of the rooting behavior of 1103 Paulsen in hydroponic systems and to evaluate the physiological and biochemical responses of cuttings under different treatments.

1. Introduction

The propagation of grapevine rootstocks is a critical phase in the process of establishing and renewing vineyards, especially in areas with intense biotic or abiotic stress. Rootstock 1103 Paulsen (Vitis berlandieri cv. Rességuier no 2 × Vitis rupestris cv. Lot) is known for its high vigor and its resistance to drought. It is also moderately tolerant to iron chlorosis and is characterized by good tolerance to high calcium levels in the soils. This rootstock is widely used in Mediterranean countries for grafting wine grape varieties [1], but despite its widespread use, the vegetative propagation process through lignified cuttings can present low rooting rates, especially when conventional rooting substrates (e.g., peat, perlite, sand) are applied [2].

In recent years, interest in the use of hydroponic systems in plant propagation has been constantly increasing [3]. Hydroponics offer a controlled rooting environment, with the possibility of modifying parameters such as temperature, humidity and oxygenation of the solution, significantly contributing to the acceleration of rooting and the production of healthy seedlings [3]. At the same time, it reduces the risk of contamination by soil-borne pathogens and eliminates the need for substrate sterilization. However, such systems present certain challenges, such as the need for continuous technical monitoring and equipment as well as the initial installation cost [3].

The incorporation of phytoregulatory substances such as indolebutyric acid (IBA), has been shown to significantly enhance rooting in many woody plant species, such as Populus spp. [4]. Recently, dopamine (L-DOPA) has attracted interest as a new, less studied phytoactive compound, with potential beneficial effects on rooting. L-DOPA belongs to phenolic compounds with strong antioxidant properties and has been shown to positively interfere with physiological processes such as germinal development, cell division, and activation of defense mechanisms [5]. In some plant species, L-DOPA has been associated with modifications in different enzymatic activities, such as phenylalanine ammonia-lyase (PAL), peroxidase (POD) and tyrosine ammonia-lyase (TAL), which are related to the formation of phenolic compounds and lignin, important elements for root development [6].

Although some studies show that L-DOPA can slow root development due to allelopathic action, others have shown a positive correlation with the enhancement of root formation under specific conditions [7]. The variation in results is attributed to the different concentrations of L-DOPA, the plant species and the environmental conditions, which makes it necessary to conduct further experiments to fully understand its mechanism of action and the possibility of its utilization in asexual propagation systems. However, research on its action on rooting is still limited and open to further investigation, especially in species such as the grapevine.

The aim of the present study was to assess the rooting ability of cuttings of rootstock 1103 Paulsen under hydroponic conditions, in relation to the application of different treatments: indolebutyric acid (IBA), dopamine (L-DOPA) and a combination of IBA and L-DOPA. At the same time, the variations in biochemical parameters of the cuttings, such as sugars, starch, phenolic compounds and antioxidants, related to rhizogenesis, were also examined.

Recording these parameters can provide a deeper understanding of the mechanisms regulating rooting and can contribute to the development of more effective vegetative propagation techniques for important grapevine rootstocks. This research aspires to enrich knowledge enhancing the application of sustainable and innovative methods in the nursery sector and contribute to the optimization of the production of healthy propagation material.

2. Materials and Methods

2.1. Plant Material and Cuttings Preparation

In this experiment, rootstock 1103 Paulsen was evaluated under hydroponic conditions with four different treatments: control, IBA, L-DOPA and a combination of IBA + L-DOPA.

The plant material was sourced from vines maintained in the Ampelographic Collection of the Laboratory of Viticulture at the Agricultural University of Athens. Leafless hardwood canes of normal vigor were selected and collected in the winter, and once the required number was collected, the cuttings were cut at 60 cm, kept in the refrigerator and, according to the legislation (KDP529/2004/No. 3853), were renewed at 45 cm before application. The dormant buds of the cuttings were removed. The cuttings comprised many nodes and internodes. The basal ends of the cuttings (approximately 5 cm of the basal part of the cutting) were then immersed in the respective treatment solutions for 24 h. The process was carried out in such a way that the basal node (the lowest node at the basal end of the cutting) was fully immersed in the treatment solution, as was the internode directly above the basal node, to ensure uniform exposure. For the phenolic, sugar and starch analyses, the basal node and the internode directly above that node were sampled.

After treatment, the cuttings were placed in small containers filled with deionized water and maintained for 60 days. All in all, 4 different treatments were performed:

• Control with zero concentration of any substance;
• 250 ppm indolebutyric acid (IBA);
• 250 ppm dopamine (L-DOPA);
• 250 ppm indolebutyric acid (IBA) and 250 ppm dopamine (L-DOPA).

For the biochemical processes and the monitoring of their variations over time, every 5 days from the moment the cuttings were placed in the containers, a sampling took place, resulting in total in 5 samplings (i.e., there was a sampling on the 1st, 5th, 10th, 15th, 30th day of the experiment) per treatment, each yielding two tissues: basal node and adjacent internode. The research followed the Completely Randomized Design with 24 replications per treatment, whereas the 5 cuttings were taken into account as 1 replication.

From each container, 20 cuttings were removed from each treatment and then the lower internodes were cut. The lowest nodes that were essentially immersed in the container were then cooled in liquid nitrogen and afterwards they were placed in bags and then in the laboratory freezer at −80 °C for their preservation until they were all collected. Then the samples were dried in a freeze dryer and ground and homogenized, forming a uniform powder. The samples were used for the measurements of phenolic compounds, starch and individual sugars.

For the determinations of the rooting measurements and overall rooting assessment (number, surface, diameter, length and presence of rooting), the sampling of 20 additional cuttings per treatment took place on the 60th day of the experiment, when the rooting process is considered completed and can be measured.

2.2. Growth Chamber Conditions

The experiment was conducted in a controlled-environment growth chamber in the laboratory as described in [8], with distilled water being the culture medium. Specifically, the temperature was set at 25.5 °C, relative humidity was regulated at 90% and light (average 600 mmol m⁻² s⁻¹) was applied by fluorescent lamps at an overhang of 100 cm above the propagation material for sixteen (16) h each day. A pump (3.5 L min⁻¹) channeled air into the water in a continuous flow and the hydroponic system’s water was renewed every 10 days.

2.3. Preparation of Solutions

2.3.1. Preparation of Solution of Indolebutyric Acid (IBA)

First, 1 g of indole-3-butyric acid (Sigma Chemical Inc., St. Louis, MO, USA) was weighed on a precision analytical scale, which was dissolved in a small amount of 50% ethanol-50% water solution, and then the volume was made up to 100 mL in a volumetric flask with the above solution (10,000 ppm). The required amount of this solution was then taken, in order to obtain a solution with a final concentration of 250 ppm, in one liter of deionized water in which the cuttings were placed for 24 h. The concentration of 250 ppm IBA was chosen based on the best results in terms of rooting percentage from previous research [8].

2.3.2. Preparation of Solution of Dopamine (L-DOPA)

First, 1 g of dopamine hydrochloride (Sigma Chemical Inc., St. Louis, MO, USA) was weighed, dissolved in a small amount of 50% ethanol-50% water solution, and then the volume was made up to 100 mL (10,000 ppm) in a volumetric flask with the above solution. The required amount of this solution was then taken, in order to obtain a solution with a final concentration of 250 ppm, in one liter of deionized water, in which the grafts were placed for 24 h.

2.3.3. Preparation of Solution of IBA + L-DOPA

First, 1 g of dopamine and 1 g of indolebutyric acid were weighed on a precision analytical balance, which were dissolved in a small amount of 50% ethanol-50% water solution and then the volume was filled with the above solution up to 100 mL in a volumetric flask. The dissolution was achieved in a beaker placed on a magnetic stirrer. The solution resulting from the above process had a concentration of 10,000 ppm. The required amount of this solution was then taken, in order to obtain a solution of 250 ppm concentration, which was added to deionized water in order to obtain 1 L of the required concentration in which the grafts were placed for 24 h.

2.4. Measurements

2.4.1. Measurements of Roots

First, 30 days from the moment of placement of the cuttings, the percentage of cuttings that had developed roots (rooting percentage %), the number of roots, the total root length (mm), the average root diameter (mm) and the total root area (mm²) per cutting were measured. The roots were scanned on a flatbed scanner (HP Scanjet G3010, HP Inc., Palo Alto, CA, USA) at 300 dpi resolution, in grayscale (8-bit), in a shallow tray of water over a black background to minimize overlap. The image analysis was obtained using the DT-software (Delta—T Scan version 2.04, Delta—T devices Ltd., Burwell, Cambridge, UK).

2.4.2. Moisture Content Determination

For the analysis, two parts of the cutting were collected: the basal node (the lowest node at the basal end of the cutting) and the directly superior internode (the internode directly above that node), to determine the fresh and dry weight of the node and internode.

Surface moisture (substrate water) was removed with absorbent paper without applying pressure. Fresh weight (FW) was immediately determined using a precision scale (Mettler AE100, Marshall Scientific, Hampton, NH, USA). Then, the node and internode were dried in an oven at 70 °C for 10 days, until they reached a constant mass, and reweighed for dry weight (DW). Moisture was calculated as follows: Moisture (% FW) = [(FW − DW)/FW] × 100.

The moisture content was used as a control for tissue hydration (i.e., absence of water stress) so that any differences in phenolics, starch and sugars are not attributed to dilutions or concentrations due to water changes but to actual metabolic processes.

2.4.3. Extraction of Phenolic Compounds

As mentioned above, two parts of the cutting were collected for phenolic analysis: the basal node (the lowest node at the basal end of the cutting) and the directly superior internode (the internode directly above that node). Initially, 200 mg of dry matter from each sample was weighed. Then, 5 mL of 70% v/v methanol acidified 1% v/v with formic acid was added. The sample was stirred and placed in a water bath at a temperature of 40 °C for 60 min. The sample was then centrifuged for 6 min at 4000 rpm and then the supernatant was separated. A second extraction of the phenolic compounds remaining in the tissue was then carried out with 70% v/v methanol acidified 1% with formic acid. The sample was stirred and placed in a water bath at a temperature of 40 °C for 60 min. The sample was again centrifuged for 6 min at 4000 rpm and the supernatant was removed so that there were 10 mL of extract in total. Then a third extraction took place on the precipitate with 70% v/v methanol acidified with 1% formic acid. The sample was then stirred and placed in a water bath at 40 °C for 60 min and then centrifuged for 6 min at 4000 rpm. The supernatant was taken to reach a total of 15 mL of extract. All fractions were combined, and the supernatants were stored at −80 °C until the analysis.

2.4.4. Determination of Phenolic Compounds and Antioxidant Activity

The determination of total phenolic compounds was performed with the Folin–Ciocalteu reagent (BDH) according to the method described by [9]. 50 μL of sample extract was added to 3.95 mL of distilled water and stirred well. 250 μL of Folin–Ciocalteu reagent was added. The sample was stirred and after 1 min, 750 μL of 20% w/v Na₂CO₃ was added. The samples were then stirred and were left to stand for 2 h. The absorbance was measured in a spectrophotometer (Hitachi U-2001 UV/Vis Spectrometer, Hitachi High-Technologies Corporation, Tokyo, Japan) at a wavelength of 760 nm. The determination of the concentrations of the samples was performed based on the reference curve of standard solutions of known catechin concentration (catechin concentration from 1000 mg L⁻¹ to 31.25 mg L⁻¹).

The total flavonoid content, total flavone content, total flavonol content and the antioxidant activity via two methods (namely the Ferric Reducing Antioxidant Power (FRAP) and the free radical scavenging activity of DPPH) were determined following the process described in [10].

2.4.5. Extraction and Determination of Individual Soluble Sugars

As mentioned above, two parts of the cutting were collected for sugar analysis: the basal node (the lowest node at the basal end of the cutting) and the directly superior internode (the internode directly above that node). The determination of the concentration of soluble sugars at the bases of the cuttings was carried out according to [11]. First, 30 mg of dry matter from each sample was weighed. Then, 2 mL of HPLC-grade water was added and the sample was stirred. The samples were placed in a microwave oven (MW) for 1.5 min at 400 W and then they were centrifuged for 5 min at 4000 rpm and the supernatant was collected. Next, 2 mL of HPLC-grade water was added to the precipitate and the above process was followed for a second time. The two supernatants were combined. Immediately following the extraction, the samples were then filtrated with a syringe filter with a pore diameter of 0.22 μmm, in order to proceed with the determination of sugars using high-performance liquid chromatography for the analysis. The analysis of the samples was performed with an HPLC pump (model 510, Waters Corporation, Milford, MA, USA), on a Hamilton Ca²⁺ column (Hamilton Company, Reno, NV, USA), at 80 °C, with a mobile phase of water and a flow rate of 0.6 mL min⁻¹. For each sample, 3 replicates were carried out. The detection of sugars was performed with a HP 1047A Refractive Index (RI) detector (SpectraLab Scientific Inc., Markham, ON, Canada)) and the processing of the chromatograms was performed using a special program on the computer (PeakSimple Chromatography Data System, Model 302, SRI Instruments, Torrance, CA, USA). The determination of the concentration of sugars was based on the reference curve of the specific sugar standards. The standard substance used was glucose, fructose and sucrose at concentrations of 1000 mg L⁻¹, 500 mg L⁻¹, 250 mg L⁻¹, 125 mg L⁻¹ and 62.5 mg L⁻¹. The verification of standard curve linearity for each sugar was as follows:

• Fructose: y = 11,225.8 · x, R² = 0.996;
• Glucose: y = 922.07 · x, R² = 0.9801;
• Sucrose: y = 1489.4 · x, R² = 0.9954.

2.4.6. Extraction and Determination of Starch

As mentioned above, two parts of the cutting were collected for starch analysis: the basal node (the lowest node at the basal end of the cutting) and the directly superior internode (the internode directly above that node). Starch determination was performed according to the enzymatic method of [11]. The procedure for starch extraction and measurement was as follows. The dry solid residue after sugar extraction was washed twice with 5 mL of 75% v/v ethanol and once with 5 mL of 100% v/v ethanol. Then, 1 mL of 0.5 N NaOH was added to the precipitate and was stirred. The samples were left for 20 min at room temperature for starch gelatinization. Next, 0.55 mL of 2 M acetic acid (CH₃COOH) was added, and the samples were centrifuged for 5 min at 4000 rpm. Then 0.5 mL of the supernatant was taken and 0.5 mL of amyloglucosidase enzyme solution (Sigma Chemical Inc., St. Louis, MO, USA) was added. The samples were then stirred and placed in a water bath for 1 h at 55 °C. Next, 0.2 mL of 1 N NaOH was added for neutralization and followed by stirring. Next, 0.5 mL of supernatant was taken to which 2 mL of glucose oxidase-peroxidase enzyme (GOD-POD reagent) from the company Biosis (GOD/PAP; Biosis, Athens, Greece) was added. The samples were placed in a water bath at 37 °C for 15 min. The color was measured with a spectrophotometer (Hitachi U-2001) at a wavelength of 510 nm. The determination of the samples was based on the reference curve of the starch concentration standards. The standard substance used was starch at concentrations of 1000 mg L⁻¹, 500 mg L⁻¹, 250 mg L⁻¹, 125 mg L⁻¹, 62.5 mg L⁻¹ and 31.25 mg L⁻¹.

2.5. Statistical Analysis

All statistical analyses were obtained using the JMP v.10 statistical software (SAS Institute Inc., Cary, NC, USA). The significance of the results was checked by analyzing the variance. The comparison of means was conducted using Tukey’s HSD method at a significance level of p ≤ 0.05. The Randomized Complete Block Design was carried out. In the presented results, means followed by different letters of the Latin alphabet indicate statistically significant differences.

3. Results

3.1. Results on the Various Measurements

Regarding the measurements performed on the roots (rooting percentage, number of roots, surface, diameter, length), the results of the present experiment revealed that the application of L-DOPA proved to be the most effective (

Table 1. Measurements of rooting percentage (%), number, area (mm²), diameter (mm) and root length (mm).

Treatments	Percentage of Rooting (%)	Number of Roots	Root Surface (mm2)	Mean Diameter of Roots (mm)	Root Length (mm)
Control	33 ± 2.5 b	9.8 ± 0.25 d	3075.65 ± 137.42 d	0.735 ± 0.018 b	3465.8 ± 32.1 c
ΙΒA	30 ± 2.7 b	19.6 ± 0.46 c	5299.86 ± 164.05 c	1.139 ± 0.051 a	4698.16 ± 207.18 b
L-DOPA	73 ± 3.1 a	44.8 ± 1.13 a	15,554.4 ± 116.20 a	1.150 ± 0.037 a	13,364.9 ± 1699.4 a
IBA + L-DOPA	76 ± 2.6 a	29.4 ± 1.59 b	9955.50 ± 341.76 b	0.844 ± 0.019 b	11,604.6 ± 288.34 a

Values are the means of quadruplicates. According to Tukey’s range test at p ≤ 0.05, mean values (Mean ± SE) in the same column assigned with different lower letters show significant differences.

), followed by the treatment with the combination of IBA + L-DOPA. Exceptions were observed in the root diameter, where the application of IBA recorded better results compared to IBA + L-DOPA, as well as in the rooting rate, where IBA + L-DOPA slightly exceeded L-DOPA, but without a statistically significant difference.

Regarding moisture content of the cuttings, at the nodes, statistically significant differences were recorded between the applications, except for control, where the values remained stable (

Table 2. Moisture content (%) in the four different treatments at the node and in the internodal space of 1103 Paulsen cuttings. The statistical analysis was performed according to the days after treatment of the experiment for the various treatments.

	Days After Treatment	Control	IBA	L-DOPA	IBA + L-DOPA
node	1	95.9 ± 0.4 a	93.9 ± 0.6 a	87.5 ± 1.1 b	94.5 ± 0.2 a
	5	96.5 ± 0.6 a	93.1 ± 0.2 b	94.0 ± 0.3 a	88.6 ± 0.7 c
	10	95.2 ± 0.4 a	91.3 ± 0.5 b	92.9 ± 0.5 ab	92.8 ± 0.6 b
	15	95.1 ± 0.1 a	94.7 ± 0.4 a	88.6 ± 0.5 b	91.2 ± 0.9 b
	30	95.1 ± 0.2 a	94.4 ± 0.4 a	91.3 ± 0.7 b	92.3 ± 0.4 b
	Days After Treatment	Control	IBA	L-DOPA	IBA + L-DOPA
internode	1	94.1 ± 0.7 a	94.7 ± 0.4 a	88.0 ± 0.1 c	90.0 ± 0.2 a
	5	94.6 ± 0.1 a	90.1 ± 0.9 b	92.1 ± 0.3 a	84.1 ± 0.2 b
	10	95.5 ± 0.3 a	89.7 ± 0.7 c	90.3 ± 0.2 b	92.9 ± 0.5 a
	15	92.1 ± 0.3 b	94.9 ± 0.6 a	85.6 ± 1.0 c	83.8 ± 0.4 b
	30	92.8 ± 0.0 b	90.6 ± 0.7 ab	89.2 ± 0.7 b	85.6 ± 0.5 b

Values are the means of quadruplicates. According to Tukey’s range test at p ≤ 0.05, mean values (Mean ± SE) in the same column assigned with different lower letters show significant differences.

). The IBA treatment resulted in a decrease in the moisture content of the cuttings until the 10th day and then it increased. The lowest moisture values were recorded in the L-DOPA and IBA + L-DOPA treatments. The cuttings with IBA exhibited a lower-value moisture content compared to the control, due to the movement of nutrients towards the root zone and as a result, it enhanced rooting. An increased rooting rate is associated with a decrease in hoarding.

Regarding L-DOPA, the highest values were recorded in all rooting parameters (percentage, length, diameter, number of roots), with a corresponding reduction in the percentage of moisture content of cutting, due to nutrient transport to the roots (Table 1 and Table 2). L-DOPA and IBA + L-DOPA presented the lowest values in the samplings.

Total phenolics displayed treatment- and tissue-specific dynamics and did not uniformly peak at the beginning of sampling (

Table 3. Concentration of total phenolics (μg catechin g⁻¹ tissue) in the node and internode space of 1103 Paulsen cuttings. Statistical analyses were performed (i) between the treatments for each day separately (columns) and (ii) between the days after treatment for each treatment separately (rows).

	Treatments	1	5	10	15	30
node	Control	10.59 ± 0.24 bA	11.59 ± 0.68 aA	10.90 ± 0.12 aA	12.26 ± 0.23 aA	11.61 ± 0.51 aA
	ΙΒA	13.37 ± 0.71 aA	12.37 ± 0.33 aAB	11.82 ± 0.33 aAB	11.11 ± 0.12 bBC	9.98 ± 0.14 aC
	L-DOPA	11.79 ± 0.08 abA	12.23 ± 0.41 aA	11.96 ± 0.20 aA	11.34 ± 0.10 bA	10.03 ± 0.40 aA
	IBA + L-DOPA	11.44 ± 0.44 abA	11.01 ± 0.38 aA	12.70 ± 0.69 aA	11.38 ± 0.26 bA	10.64 ± 0.64 aA
	Treatments	1	5	10	15	30
internode	Control	10.25 ± 0.42 aB	11.37 ± 0.80 aAB	10.45 ± 0.57 aB	10.17 ± 0.16 aB	17.23 ± 2.80 aA
	ΙΒA	10.56 ± 0.43 aA	10.44 ± 0.22 aA	10.55 ± 0.31 aA	10.96 ± 1.00 aA	9.28 ± 0.29 bA
	L-DOPA	10.10 ± 0.17 aA	11.14 ± 0.28 aA	11.90 ± 0.22 aA	11.11 ± 0.58 aA	10.32 ± 0.94 bA
	IBA + L-DOPA	10.43 ± 0.33 aA	11.72 ± 0.54 aA	11.29 ± 0.16 aA	11.15 ± 0.15 aA	10.86 ± 0.28 abA

Values are the means of quadruplicates. According to Tukey’s range test at p ≤ 0.05, mean values (Mean ± SE) in the same column assigned with different lower letters indicate significant statistical difference between the treatments for each day separately, and in the same row assigned with different capital letters indicate significant statistical difference between the days after treatment for each treatment separately.

). In the node, the IBA treatment exhibited higher concentration and declined progressively thereafter, whereas in the internode IBA values remained relatively stable before declining towards the end; by contrast, in the internode, control treatment tended to increase towards the end of the sampling period, while L-DOPA and IBA + L-DOPA showed intermediate fluctuations without a consistent early peak. More broadly, polyphenols often accumulate when growth slows, particularly phenylpropanoids that serve as lignin precursors prior to cell wall incorporation [12]. However, early declines at the base of cuttings have also been documented (e.g., cherry) [13], despite the expectation of a transient wound-induced rise [14]. Reports for shoots are mixed, reflecting tissue specificity and timing (see also bean cuttings) [15].

Flavonoids showed treatment- and tissue-specific patterns (

Table 4. Total flavonoid concentration (μg catechin g⁻¹ tissue) in the node and internode space of 1103 Paulsen cuttings. Statistical analyses were performed (i) between the treatments for each day separately (columns) and (ii) between the days after treatment for each treatment separately (rows).

	Treatments	1	5	10	15	30
node	Control	19.96 ± 0.34 bB	20.70 ± 0.87 abB	23.05 ± 0.19 aA	23.60 ± 0.28 aA	21.96 ± 0.16 abAB
	ΙΒA	21.44 ± 0.85 abB	22.56 ± 0.75 aA	18.00 ± 0.35 cC	19.19 ± 0.34 cBC	18.71 ± 0.60 bBC
	L-DOPA	19.70 ± 0.37 bB	20.24 ± 0.47 abB	23.33 ± 0.60 aA	21.46 ± 0.17 bAB	20.13 ± 0.67 abB
	IBA + L-DOPA	23.60 ± 0.73 aA	17.82 ± 1.68 bB	20.80 ± 0.27 bAB	23.76 ± 0.11 aA	22.54 ± 1.29 aA
	Treatments	1	5	10	15	30
internode	Control	18.90 ± 0.81 aA	16.12 ± 0.73 bB	15.42 ± 0.50 bB	19.22 ± 0.56 abA	20.28 ± 0.05 aA
	ΙΒA	15.87 ± 0.48 aBC	19.58 ± 0.89 aAB	16.38 ± 1.70 bABC	20.40 ± 0.50 abA	15.04 ± 0.37 cC
	L-DOPA	18.65 ± 1.02 aAB	18.88 ± 0.38 abAB	20.71 ± 0.27 aA	20.80 ± 1.07 aA	17.11 ± 0.18 bB
	IBA + L-DOPA	17.84 ± 0.69 aA	17.48 ± 0.40 abA	19.48 ± 0.48 abA	17.46 ± 0.43 bA	18.20 ± 0.41 bA

Values are the means of quadruplicates. According to Tukey’s range test at p ≤ 0.05, mean values (Mean ± SE) in the same column assigned with different lower letters indicate significant statistical difference between the treatments for each day separately, and in the same row assigned with different capital letters indicate significant statistical difference between the days after treatment for each treatment separately.

). In the node, significant contrasts were detected on several days, but not universally among all 4 treatments. On day 1, IBA + L-DOPA treatment exhibited higher concentrations than control and L-DOPA; on day 5, IBA recorded the highest flavonoids concentration, while IBA + L-DOPA the lowest; on days 10–15, control and IBA + L-DOPA treatments showed higher concentrations compared to IBA; on day 30, IBA + L-DOPA treatment recorded the highest flavonoids concentration, whereas IBA the lowest. Regarding the differences between the treatments over time, control peaked at days 10–15 compared to days 1–5. IBA peaked on day 5 and then declined, while L-DOPA treatment peaked on day 10. IBA + L-DOPA treatment showed a U-shaped profile with a decrease on day 5 and a subsequent increase. In the internode, between treatments per day, on day 1 there were no significant differences among the treatments. On day 5, IBA treatment exhibited higher concentrations compared to control, while between days 10–15 L-DOPA recorded higher concentrations when compared to control and IBA treatments. On day 30, control exhibited the highest concentration and IBA the lowest. Regarding the differences within treatments, flavonoid concentration increased in control on day 30, compared to days 5–10. In IBA treatment, flavonoid concentration decreased in the node up until day 30. L-DOPA treatment resulted in an increase up to days 10–15 and then decreased, while in IBA + L-DOPA treatment flavonoid concentration remained relatively stable over time.

To sum up, significant effects are day- and treatment-specific rather than universal: IBA treatment tends to result in lower concentrations both in the nodes and internodes, while IBA + L-DOPA treatment exhibits higher concentrations in the node.

In the nodal flavones, statistically significant differences are observed between the samples for all treatments, except for the control, where the values remain stable (

Table 5. Total flavone and flavonol concentrations (μg rutin g⁻¹ tissue) in the node and internode space of 1103 Paulsen cuttings. Statistical analyses were performed (i) between the treatments for each day separately (columns) and (ii) between the days after treatment for each treatment separately (rows).

	Treatments	1	5	10	15	30
node	Control	0.29 ± 0.01 bA	0.46 ± 0.09 bA	0.28 ± 0.02 bA	0.40 ± 0.01 bA	0.47 ± 0.05 bA
	ΙΒA	0.17 ± 0.01 cC	0.20 ± 0.03 cC	0.61 ± 0.08 aB	0.55 ± 0.02 aB	1.10 ± 0.14 aA
	L-DOPA	0.47 ± 0.03 aA	0.33 ± 0.01 bcB	0.38 ± 0.03 bAB	0.35 ± 0.03 bAB	0.36 ± 0.04 bAB
	IBA + L-DOPA	0.26 ± 0.01 bB	0.72 ± 0.05 aA	0.31 ± 0.02 bB	0.24 ± 0.01 cB	0.32 ± 0.03 bB
	Treatments	1	5	10	15	30
internode	Control	0.21 ± 0.02 bC	0.43 ± 0.03 aAB	0.53 ± 0.04 aA	0.33 ± 0.03 bBC	0.46 ± 0.01 bAB
	ΙΒA	0.64 ± 0.05 aA	0.27 ± 0.03 bB	0.65 ± 0.01 aA	0.32 ± 0.002 bB	0.69 ± 0.05 aA
	L-DOPA	0.24 ± 0.04 bB	0.36 ± 0.02 abAB	0.36 ± 0.02 bAB	0.31 ± 0.04 bAB	0.39 ± 0.02 bA
	IBA + L-DOPA	0.20 ± 0.09 bB	0.47 ± 0.05 aA	0.34 ± 0.03 bAB	0.53 ± 0.01 aA	0.41 ± 0.02 bAB

Values are the means of quadruplicates. According to Tukey’s range test at p ≤ 0.05, mean values (Mean ± SE) in the same column assigned with different lower letters indicate significant statistical difference between the treatments for each day separately, and in the same row assigned with different capital letters indicate significant statistical difference between the days after treatment for each treatment separately.

). In L-DOPA and in the combined application with IBA + L-DOPA, a decreasing concentration trend is recorded, while in IBA treatment, the concentration gradually increases, being the only treatment with an upward trend. In the internode, statistically significant differences are recorded in all treatments. The concentrations exhibit fluctuations throughout the experiment, without a clear or stable trend in any of the treatments.

Regarding flavanols in the node, statistically significant differences are observed in the control and in the IBA treatment, in which a decrease in concentration is recorded, particularly on day 30 in IBA (

Table 6. Total flavanol concentration (μg catechin g⁻¹ tissue) in the node and internode space of 1103 Paulsen cuttings. Statistical analyses were performed (i) between the treatments for each day separately (columns) and (ii) between the days after treatment for each treatment separately (rows).

	Treatments	1	5	10	15	30
node	Control	15.45 ± 0.83 aAB	13.70 ± 0.60 aB	17.62 ± 1.02 aA	14.79 ± 0.49 aAB	13.61 ± 0.48 aB
	ΙΒA	15.40 ± 0.78 aA	12.54 ± 0.19 aB	11.56 ± 0.19 bB	12.99 ± 0.81 abAB	8.61 ± 0.49 cC
	L-DOPA	10.47 ± 0.72 baA	12.18 ± 0.83 aA	12.82 ± 0.69 bA	11.09 ± 0.13 bA	11.87 ± 0.21 bA
	IBA + L-DOPA	12.84 ± 0.92 abA	12.85 ± 0.44 aA	10.84 ± 0.54 bA	12.63 ± 1.25 abA	11.33 ± 0.26 bA
	Treatments	1	5	10	15	30
internode	Control	13.07 ± 0.46 aA	9.44 ± 0.92 aB	12.64 ± 0.95 aAB	13.64 ± 0.94 aA	10.85 ± 0.24 bAB
	ΙΒA	11.68 ± 0.57 abA	10.45 ± 1.18 aA	11.36 ± 0.56 aA	12.46 ± 0.71 abA	7.09 ± 0.20 dB
	L-DOPA	9.17 ± 0.37 cB	10.11 ± 0.28 aAB	11.79 ± 0.51 aAB	11.36 ± 1.11 abAB	12.20 ± 0.35 aA
	IBA + L-DOPA	10.70 ± 0.62 bcA	10.15 ± 0.18 aA	10.56 ± 0.45 aA	8.71 ± 0.79 bB	9.15 ± 0.05 cA

Values are the means of quadruplicates. According to Tukey’s range test at p ≤ 0.05, mean values (Mean ± SE) in the same column assigned with different lower letters indicate significant statistical difference between the treatments for each day separately, and in the same row assigned with different capital letters indicate significant statistical difference between the days after treatment for each treatment separately.

). In contrast, in the L-DOPA and IBA + L-DOPA treatments, the values remain relatively stable, without significant fluctuations. In the internode, significant differences are observed in all treatments, except for treatment IBA + L-DOPA. In the control and IBA treatments, a decrease in flavanols is recorded, while in L-DOPA treatment an increase in concentration is observed up to day 30.

Regarding the antioxidant activity determined via the FRAP method, in the node, statistically significant differences are observed in all treatments (

Table 7. Antioxidant capacity (μg Trolox g⁻¹ tissue) using the FRAP method in the node and internode space of 1103 Paulsen cuttings. Statistical analyses were performed (i) between the treatments for each day separately (columns) and (ii) between the days after treatment for each treatment separately (rows).

	Treatments	1	5	10	15	30
node	Control	43.82 ± 0.81 dD	59.31 ± 0.42 aB	69.79 ± 0.48 aA	54.69 ± 0.49 aC	54.01 ± 0.32 bC
	ΙΒA	59.94 ± 1.90 bA	47.55 ± 0.68 cB	58.55 ± 1.33 bA	47.65 ± 1.50 bB	49.61 ± 0.38 cB
	L-DOPA	50.15 ± 0.91 cAB	49.92 ± 0.79 bcAB	53.61 ± 0.48 bA	52.34 ± 0.58 abA	47.65 ± 1.20 cB
	IBA + L-DOPA	75.13 ± 0.64 aB	52.26 ± 1.32 bC	45.21 ± 1.91 cD	50.65 ± 1.62 abCD	94.31 ± 0.51 aA
	Treatments	1	5	10	15	30
internode	Control	72.56 ± 2.66 aD	42.29 ± 0.31 cB	55.02 ± 0.31 bA	47.15 ± 7.00 bC	71.00 ± 2.62 aC
	ΙΒA	52.60 ± 0.46 bB	49.26 ± 0.48 bC	53.66 ± 0.17 bB	75.13 ± 1.12 aA	43.16 ± 0.52 cD
	L-DOPA	56.11 ± 2.02 bBC	59.85 ± 1.53 aAB	66.50 ± 1.10 aA	52.82 ± 1.43 bC	44.23 ± 1.22 cD
	IBA + L-DOPA	60.44 ± 0.90 bAB	52.23 ± 1.75 bBC	66.32 ± 1.06 aA	46.58 ± 3.51 bC	52.82 ± 1.27 bBC

Values are the means of quadruplicates. According to Tukey’s range test at p ≤ 0.05, mean values (Mean ± SE) in the same column assigned with different lower letters indicate significant statistical difference between the treatments for each day separately, and in the same row assigned with different capital letters indicate significant statistical difference between the days after treatment for each treatment separately.

). The control generally follows an increasing trend, peaking on day 30, while IBA and L-DOPA show fluctuations, ending in values similar to or higher than day 1. Treatment IBA + L-DOPA shows a decrease after day 15, despite the sharp intermediate increase. Correspondingly, in the internode, significant differences are recorded in all treatments. The IBA and L-DOPA treatments show fluctuations with final values similar to the initial day, while the control and IBA + L-DOPA show a clear decrease until the end, with the latter showing the lowest value of all.

Regarding the DPPH method, all treatments in the node show statistically significant fluctuations (

Table 8. Antioxidant capacity (μg Trolox g⁻¹ tissue) using the DPPH method in the node and internode space of 1103 Paulsen cuttings. Statistical analyses were performed (i) between the treatments for each day separately (columns) and (ii) between the days after treatment for each treatment separately (rows).

	Treatments	1	5	10	15	30
node	Control	11.82 ± 0.29 bC	15.97 ± 0.19 aB	17.90 ± 0.10 aA	15.00 ± 0.35 aB	19.00 ± 0.44 aA
	ΙΒA	15.44 ± 0.17 aA	12.68 ± 0.41 bC	14.41 ± 0.19 cAB	13.92 ± 0.46 bBC	15.10 ± 0.19 cAB
	L-DOPA	15.32 ± 0.05 aB	15.62 ± 0.12 aB	13.26 ± 0.23 dC	14.13 ± 0.32 bC	17.42 ± 0.24 bA
	IBA + L-DOPA	15.60 ± 0.004 aB	15.76 ± 0.19 aB	15.35 ± 0.19 bB	17.59 ± 0.27 aA	15.38 ± 0.22 cB
	Treatments	1	5	10	15	30
internode	Control	14.92 ± 0.66 aA	16.23 ± 0.11 bA	15.98 ± 0.19 aA	14.73 ± 0.07 cA	11.94 ± 0.22 bB
	ΙΒA	15.94 ± 0.08 aAB	14.03 ± 0.41 cC	16.43 ± 0.25 aA	15.54 ± 0.36 bcAB	14.76 ± 0.33 aBC
	L-DOPA	14.51 ± 0.08 aB	19.00 ± 0.30 aA	13.15 ± 0.60 bB	19.00 ± 0.16 aA	14.19 ± 0.08 aB
	IBA + L-DOPA	14.95 ± 0.49 aB	15.31 ± 0.14 bAB	13.60 ± 0.08 bC	16.31 ± 0.13 bA	8.39 ± 0.10 cD

Values are the means of quadruplicates. According to Tukey’s range test at p ≤ 0.05, mean values (Mean ± SE) in the same column assigned with different lower letters indicate significant statistical difference between the treatments for each day separately, and in the same row assigned with different capital letters indicate significant statistical difference between the days after treatment for each treatment separately.

). IBA and L-DOPA treatments show lower values on day 30, while IBA + L-DOPA treatment shows a decrease between days 5–15 and a sharp increase on day 30. The control increases until day 10 and then decreases. In the internode, fluctuations are also recorded, with maximum values on days 10–15, except for the control, which peaks on day 30.

Overall, antioxidant compounds, as measured by both DPPH and FRAP, do not follow a consistent pattern (Table 7 and Table 8). Fluctuations are observed in all treatments, but a general trend is that the values on day 30 are lower than on day 1. Exceptions are the control and L-DOPA treatments in DPPH, and the control and IBA + L-DOPA treatments in FRAP. After cutting, the cuttings enter a state of stress due to lack of water and nutrients. L-DOPA does not show a consistent pattern, while IBA + L-DOPA treatment presents in some cases unreasonably high values.

The variation in starch concentration in the nodes and internodes of rootstock 1103 Paulsen under the different treatments is presented in

Table 9. Starch concentration (μg g⁻¹ tissue) in the node and internode space of 1103 Paulsen cuttings. Statistical analyses were performed (i) between the treatments for each day separately (columns) and (ii) between the days after treatment for each treatment separately (rows).

	Treatments	1	5	10	15	30
node	Control	5.69 ± 0.22 bAB	6.33 ± 0.18 bA	4.93 ± 0.06 bB	5.47 ± 0.21 cB	5.25 ± 0.08 bB
	ΙΒA	7.35 ± 0.12 aA	5.59 ± 0.19 cB	3.68 ± 0.04 cC	5.93 ± 0.12 bcB	6.04 ± 0.09 aB
	L-DOPA	6.81 ± 0.08 aA	6.31 ± 0.09 bB	5.78 ± 0.13 aC	6.34 ± 0.03 abB	5.36 ± 0.03 bD
	IBA + L-DOPA	6.12 ± 0.03 bBC	7.35 ± 0.08 aA	5.55 ± 0.19 aC	6.66 ± 0.12 aAB	5.57 ± 0.25 abC
	Treatments	1	5	10	15	30
internode	Control	6.45 ± 0.13 abA	6.23 ± 0.08 aAB	5.83 ± 0.12 aB	5.72 ± 0.14 bB	4.61 ± 0.13 bC
	ΙΒA	6.56 ± 0.11 aA	4.91 ± 0.09 bC	5.93 ± 0.04 aB	6.57 ± 0.21 aA	5.83 ± 0.15 aB
	L-DOPA	5.86 ± 0.08 bA	4.81 ± 0.10 bC	4.35 ± 0.05 cD	5.26 ± 0.07 bB	4.09 ± 0.08 bD
	IBA + L-DOPA	6.36 ± 0.24 abA	6.47 ± 0.11 aA	5.12 ± 0.12 bB	5.18 ± 0.09 bB	6.37 ± 0.22 aA

Values are the means of quadruplicates. According to Tukey’s range test at p ≤ 0.05, mean values (Mean ± SE) in the same column assigned with different lower letters indicate significant statistical difference between the treatments for each day separately, and in the same row assigned with different capital letters indicate significant statistical difference between the days after treatment for each treatment separately.

. Starch dynamics depended on treatment, time and tissue (Treatment × Time interaction). In the basal node, auxin-containing treatments (IBA, IBA + L-DOPA) showed an early decrease in starch relative to day 1, consistent with mobilization of reserves at the rooting site. Control and L-DOPA displayed medium fluctuations. In the adjacent internode, starch changes were smaller and often delayed compared to the node. These patterns align with the physiology of leafless, dormant cuttings, where early growth relies on stored carbohydrates and auxin re-partitions carbon toward callus and adventitious root initiation.

The concentrations of individual sugars in the nodes and internodes of rootstock 1103 Paulsen under the different treatments are presented in

Table 10. Glucose, fructose and sucrose concentration (μg g⁻¹ tissue) in the node and internode space of 1103 Paulsen cuttings. Statistical analyses were performed (i) between the treatments for each day separately (columns) and (ii) between the days after treatment for each treatment separately (rows).

Treatments

1

5

10

15

30

Fructose

Glucose

Sucrose

Fructose

Glucose

Sucrose

Fructose

Glucose

Sucrose

Fructose

Glucose

Sucrose

Fructose

Glucose

Sucrose

node

Control

13.52 ± 0.10 bA

15.51 ± 0.86 aA

33.26 ± 1.15 aA

8.28 ± 0.13 aC

8.18 ± 0.12 aB

14.41 ± 0.22 aB

10.40 ± 0.37 aB

7.45 ± 0.13 aB

13.72 ± 0.55 aBC

5.66 ± 0.09 aD

6.26 ± 0.44 aB

11.03 ± 0.05 aC

3.45 ± 0.07 bE

2.93 ± 0.08 aC

5.98 ± 0.30 bD

ΙΒA

9.40 ± 0.05 cA

9.43 ± 0.24 bA

22.29 ± 1.05 bA

7.60 ± 0.11 abB

5.66 ± 0.19 bcB

7.29 ± 0.11 cC

5.60 ± 0.11 bC

4.93 ± 0.10 bB

11.04 ± 0.28 bB

4.37 ± 0.09 bcD

4.76 ± 0.14 bC

6.91 ± 0.07 dC

2.77 ± 0.10 cE

3.14 ± 0.24 aD

4.01 ± 0.16 cD

L-DOPA

8.57 ± 0.11 cA

9.26 ± 0.12 bA

17.99 ± 0.56 cA

7.22 ± 0.28 bB

6.26 ± 0.14 bB

14.59 ± 0.14 aB

4.86 ± 0.27 bC

3.50 ± 0.28 cC

8.64 ± 030 cD

4.77 ± 0.24 bC

3.89 ± 0.17 bcC

9.51 ± 0.35 bD

7.15 ± 0.24 aB

2.16 ± 0.09 bD

11.82 ± 0.23 aC

IBA + L-DOPA

15.31 ± 0.45 aA

9.73 ± 0.23 bA

25.50 ± 0.87 bA

4.94 ± 0.04 cB

5.52 ± 0.17 cB

9.74 ± 0.15 bB

5.51 ± 0.09 bB

5.03 ± 0.04 bB

8.87 ± 0.19 cB

3.82 ± 0.12 cC

2.78 ± 0.17 cC

8.12 ± 0.29 cB

2.37 ± 0.08 cD

3.04 ± 0.11 aC

5.82 ± 0.24 bC

Treatments

1

5

10

15

30

Fructose

Glucose

Sucrose

Fructose

Glucose

Sucrose

Fructose

Glucose

Sucrose

Fructose

Glucose

Sucrose

Fructose

Glucose

Sucrose

internode

Control

20.40 ± 0.34 aA

15.64 ± 0.09 aA

42.48 ± 0.26 aA

11.07 ± 0.11 aC

11.42 ± 0.26 aB

19.85 ± 0.55 aC

13.11 ± 0.09 aB

10.24 ± 0.13 aC

21.36 ± 0.52 aC

8.87 ± 0.08 aD

9.37 ± 0.40 aC

34.57 ± 0.22 aB

5.04 ± 0.11 aE

4.07 ± 0.29 bD

11.09 ± 0.51 bD

ΙΒA

8.72 ± 0.11 cΒ

7.82 ± 0.27 cA

29.48 ± 0.41 cA

10.03 ± 0.23 bA

8.13 ± 0.27 bA

19.14 ± 0.79 aB

4.30 ± 0.11 cD

5.08 ± 0.05 bB

9.79 ± 0.14 cD

5.13 ± 0.07 bC

4.38 ± 0.17 cBC

13.29 ± 0.27 bC

4.03 ± 0.10 cD

3.86 ± 0.02 bC

13.60 ± 0.03 aC

L-DOPA

7.94 ± 0.19 cΒ

7.67 ± 0.30 cA

29.56 ± 0.68 cA

6.83 ± 0.15 cC

6.85 ± 0.13 cB

13.89 ± 0.25 bB

8.58 ± 0.18 bAB

5.40 ± 0.01 bC

13.62 ± 0.13 bB

9.10 ± 0.22 aA

7.55 ± 0.05 bAB

14.09 ± 0.16 bB

4.60 ± 0.15 abA

5.16 ± 0.20 aC

10.24 ± 0.25 bC

IBA + L-DOPA

14.50 ± 0.31 bA

11.50 ± 0.08 bA

36.61 ± 0.16 bA

9.69 ± 0.23 bB

6.65 ± 0.26 cB

15.75 ± 0.13 bB

2.53 ± 0.12 dD

2.17 ± 0.12 cD

8.22 ± 0.27 dE

3.92 ± 0.14 cC

4.56 ± 0.24 cC

13.87 ± 0.40 bC

4.46 ± 0.04 bcC

4.11 ± 0.04 bC

11.58 ± 0.45 bD

Values are the means of quadruplicates. According to Tukey’s range test at p ≤ 0.05, mean values (Mean ± SE) in the same column assigned with different lower letters indicate significant statistical difference between the treatments for each day separately, and in the same row assigned with different capital letters indicate significant statistical difference between the days after treatment for each treatment separately.

. Regarding the nodes, statistically significant differences are recorded in all treatments, with a generally decreasing course of concentrations. The lowest values appear on day 30 in the control and in L-DOPA, with a statistically significant difference, and with a significant decrease already from day 10. In the internode space, a corresponding trend is observed, with the exception of the treatment with L-DOPA (increase on day 15) and IBA + L-DOPA, which shows the lowest value on day 10, again with a statistically significant difference compared to the other treatments. Higher concentrations are recorded in the control in the node and internode.

Regarding fructose concentration, in the node, most treatments show a decreasing trend, until day 10. L-DOPA varies, with a slight increase on day 30. The IBA and IBA + L-DOPA treatments show the lowest values with a statistically significant difference. In the internode, all treatments exhibit statistically significant differences, with fluctuations at different time points: between the 5th–15th day in the control, IBA and L-DOPA, while in IBA + L-DOPA treatment a change is observed between the 15th–30th day.

Regarding glucose concentration, statistically significant differences are observed in all treatments. In the node, the control and IBA + L-DOPA follow a decreasing course, while in IBA and L-DOPA, fluctuations appear (change mainly on the 10th and 5th day, respectively). In the internode, all treatments exhibit statistically significant differences, with L-DOPA showing a gradual decrease, while the other treatments exhibit fluctuations. The lowest value is observed on the 10th day in IBA + L-DOPA treatment.

Regarding sucrose concentration, in the node, the control and IBA + L-DOPA treatments show a steady decline, while IBA and L-DOPA show fluctuations. In the internode, the values gradually decrease in L-DOPA, while the remaining treatments show fluctuations with a minimum value on the 10th day.

3.2. Principal Component Analysis (PCA)

PCA transforms an original data set, all measurements included, into a smaller set of uncorrelated new variables (Principal Components, where eigenvalues are bigger than 1). The PCA was carried out on the measurements under study. It produced 4 components, in declining order of importance. Those 4 components accounted for and explained 72.14% of the total variability between and among the different measurements (

Table 11. Principal components (PC) of the variables evaluated.

Principal Components
1	2	3	4
%Contribution to variability
29.52	21.57	12.88	8.16
Eigenvalue
4.42	3.33	1.93	1.23
Related measurements
Rooting percentage	Fructose	Total flavonoids	Total Flavanols
Root number	Glucose	Total phenols	DPPH
Root surface	Sucrose	FRAP	Starch
Mean diameter of roots		Total Flavones and Flavonols

). All measurements grouped under the same principal component show a strong correlation between and among them.

The first principal component (PC1) accounted for 29.52% of the total variability. It was defined by the following measurements: rooting percentage, root number, root surface, and mean diameter of roots. The second principal component (PC2) explained another 21.57% of the total variability. PC2 was defined by the individual sugars, fructose, glucose and sucrose. The third principal component (PC3) explained another 12.88% of the total variability and was defined by total flavonoids, total phenols, total flavones and flavonols, and antioxidant capacity as measured with the FRAP method (Table 11, Figure 1).

The PCA plot illustrates the distribution of various grape-related parameters across the first two principal components, which explain 29.5% and 21.6% of the total variance, respectively (Figure 1). Key traits such as rooting percentage, root number, root surface, and mean diameter of roots appear highly correlated along Component 1. Component 2 captures variability associated with individual sugars.

3.3. Effect and Interaction of Variability Factors

Three variability factors were considered: Days after treatment, Part of cane and Treatment. They all significantly influenced the phenolic content, antioxidant capacity and individual sugar content of cuttings (p ≤ 0.001). It should be noted that the three variability factors showed no significance in the measurements of roots (rooting percentage, root number, root surface, mean diameter of roots and total length of roots), since these measurements took place on the last day, and not during the days after treatment (

Table 12. Effect of variability factors (A: Days after treatment, B: part of cane, C: treatment) on the phenolic content, antioxidant capacity and individual sugar content of 1103 Paulsen cuttings.

	Total Flavonoids	Total Flavones and Flavonols	Total Phenols	Total Flavanols	DPPH	FRAP	Starch	Fructose	Glucose	Sucrose
Factors of Variability	Significance level	Significance level	Significance level	Significance level	Significance level	Significance level	Significance level	Significance level	Significance level	Significance level
A	***	***	**	***	***	***	***	***	***	***
B	***	ns	**	***	***	***	***	***	***	***
C	***	***	ns	***	***	***	***	***	***	***
Interactions
AB	ns	**	***	***	***	***	***	***	***	***
AC	***	***	***	***	***	***	***	***	***	***
BC	**	ns	*	**	***	***	***	***	***	***
ABC	***	***	**	*	***	***	***	***	***	***

Single-factorial, two-factorial, and multiparametric analysis of the mean concentrations of groups of phenolic compounds, antioxidant capacity and individual sugar content in cuttings. (*), (**), (***): significant at p ≤ 0.05, p ≤ 0.01, p ≤ 0.001. ns: not significant.

).

4. Discussion

The application with L-DOPA and IBA + L-DOPA showed high values in rooting percentage, diameter and length of roots, exhibiting better results compared to the IBA-treated cuttings. While positive effects on rooting have been reported in previous research [16] and on different species, they have been obtained with higher IBA concentrations: a positive effect of IBA (3000–4000 mg L⁻¹) was reported on rose [17], while at 2000 ppm, high percentage of rooting was observed in Grewia asiatica L. cuttings [18]. In a greenhouse, for Vitis vinifera, optimal rates at a concentration of IBA 4000 ppm were recorded [19], while optimal rates at 1000 ppm were recorded for rootstock 1103 Paulsen [20]. At the same time, the current experiment showed effectiveness at lower concentrations, since the IBA concentration of 250 ppm was chosen and used in this experiment based on the good rooting results found in previous research [8]. Auxin is active at certain levels, while at higher concentrations it may inhibit root formation [19]. The action of IBA is related to cambial activation and nutrient transport and it is naturally produced in shoots and leaves, while its synthetic form prevents dehydration [21].

In addition, high carbohydrates and low nitrogen enhance root formation [18], while plants with easy rooting require lower concentrations of IBA [8]. The number of roots is related to carbohydrate hydrolysis and amino acid accumulation, while root length is related to nutrient transport and cell elongation [22]. The applications of L-DOPA and IBA + L-DOPA gave very good results, with the former being superior. The use of L-DOPA has not been extensively studied in woody cuttings. It has been reported to have an allelochemical effect, negatively affecting Gramineae, Leguminosae, and to a lesser extent Brassicaceae and Cucurbitaceae [23]. In soybean, it reduced root growth by up to 62.4% at a L-DOPA concentration of 99 ppm [6]. A strong inhibition of root growth was recorded in several species [23]. Nevertheless, in the present experiment, a positive effect was observed, probably due to different plant material (lignified cuttings), which requires further study.

The treatments with L-DOPA and IBA + L-DOPA exhibited the highest values in all rooting parameters, with a corresponding reduction in the percentage of moisture content of cutting, due to nutrient transport to the roots. In the present study, the moisture content of the cuttings was used as a control for tissue hydration (i.e., absence of water stress) so that any differences observed in phenolics, starch and sugars are not attributed to dilutions or concentrations due to water changes, but to actual metabolic processes. The moisture values remained relative stable throughout the experiment confirming the hypothesis under study.

Total phenolics displayed treatment- and tissue-specific dynamics and did not uniformly peak at the beginning of sampling. L-DOPA treatment shows increased concentration in the initial days of the experiment (days 1–5), which is in agreement with literature, as rhizogenesis favors the synthesis of polyphenols, mainly lignin precursors [12]. A decrease in phenolics was observed from the 1st day in cherry cuttings [13], despite the expected increase due to traumatic stress [14]. A similar increase was recorded in beans [15], and in pome fruits on the 2nd day [24].

The phenolic subgroups followed a similar trend, with the following exceptions: flavanols in the node and internode (IBA), and flavonoids of the control in the node showed low initial values that increased towards the end. The course of phenolics in the present experiment is initially increasing and decreases with the onset of rooting, which depends on the applied treatment. The cuttings with IBA and IBA + L-DOPA show a faster decrease due to early rooting. IBA treatment led to a steady decrease in phenolics, with the lowest values on the 30th day in the node and internode, a result that agrees with a previous study [15].

The relationship between phenolic content and rooting has been highlighted [13]. The decrease in phenolics coincided with the onset of rooting and with a reduction in class III peroxidase (EC 1.11.1.7) activity, an enzyme system historically referred to as ‘IAA-oxidase activity’ because these peroxidases catalyze the H₂O₂-dependent oxidation of IAA [25]. It was observed that in explants with IBA or polyamines, phenolic compounds showed fluctuations with generally higher values than the control [15], an observation that is also confirmed in the present experiment. In L-DOPA and IBA + L-DOPA treatments, phenolic compounds peak before day 15 and then decrease, while in the control they exhibit an increase. In an experiment in soybean, L-DOPA inhibited root growth and increased tyrosine, phenylalanine and lignin, activating enzymes such as PAL and POD, indirectly enhancing phenolic compounds [6]. Although the L-DOPA-phenolic relationship is confirmed, the degree of the effect remains unclear. In all cases, phenolic concentrations are higher in the node than in the internode. Vascular differentiation begins in the nodes, possibly due to increased auxin concentration and subsequent phenolic activity [26]. Phenolics also protect IAA from oxidation [14], which reinforces their importance in successful rooting.

L-DOPA exhibits a concentration-dependent dual role: at low concentrations, in the presence of Fe³⁺/Fe²⁺ and H₂O₂, it enhances •OH generation (Fenton cycling), whereas at higher concentrations it suppresses radical formation via scavenging/chelation [27]. In addition, antioxidant activity has been recorded in various in vitro methods, such as DPPH reduction and lipid peroxidation inhibition [28]. Finally, the higher concentration of antioxidants in the nodes compared to the internodes can probably be attributed to vascular differentiation and more intense auxin action in the area [26].

Carbohydrates are considered key indicators as they are the products of photosynthesis and a major source of energy as well as structural components for the formation of root archetypes [29]. They have been reported to contribute to rooting not only as energy substrates, but also through a positive correlation with rooting capacity [30].

In the present study, glucose and fructose concentrations followed a decreasing trend, more intensive in the node, which is related to their being consumed during rhizogenesis. Sucrose also decreased, with the greatest decrease recorded in the cuttings immersed in IBA, which showed the lowest values at the end of the experiment. This is in agreement with studies reporting the movement and consumption of sugars in the rooting zone to meet energy requirements [31]. On the contrary, some studies argue that soluble sugars may increase again after the initial phase of root formation, due to increased photosynthesis or starch degradation [32]. In the current experiment, sugar concentrations gradually decreased without a clear second increase, which is probably associated with limited photosynthetic activity or reduced sugar transport to the measurement points.

Starch concentration showed smaller fluctuations compared to sugars, confirming its less direct participation in rooting. In cuttings treated with IBA, higher starch values were recorded at the beginning, which then decreased, an element also supported by [33]. IBA treatment led to a higher consumption of soluble sugars, probably due to enhanced basipetal transport and activation of rooting metabolic pathways. The lower concentration of sugars at day 30 in IBA samples indicates their increased use for root formation, strengthening the hypothesis of their correlation with rooting success [34]. Regarding L-DOPA, intermediate sugar concentrations were observed between control and IBA. Although catecholamines have not been extensively studied in cuttings, there are indications that they affect sugar metabolism [35], which requires further investigation.

5. Conclusions

The hydroponic system provides excellent rooting results as long as the water has sufficient oxygenation. The cuttings immersed in L-DOPA treatment recorded the best results both in terms of the rooting rate as well as in the remaining root measurements, followed by the IBA + L-DOPA and IBA treatments. However, experimentation is needed with other L-DOPA concentrations as well as with lignified cuttings of other rootstocks.

Regarding the biochemical activity of the cuttings, during the experiment, it was observed that the phenolic compounds as well as the individual sugars and starch began to decrease from the moment the first roots started to appear, which is considered normal since they participate indirectly in the rhizogenesis process of the cuttings. It should also be noted that the more intensive the rhizogenesis process, the more intensive the decrease in the various biochemical compounds.