Evaluation of Shoot Collection Timing and Hormonal Treatment on Seedling Rooting and Growth in Four Poplar Genomic Groups

Abstract

Populus spp. is an economically valuable tree worldwide, known for its adaptability, fast growth, and versatile wood, often cultivated in short-rotation plantations. Effective propagation is crucial for rapid genetic improvement and global demand for forest products and biomass energy. This study focused on the rooting and growth of poplar cuttings, examining shoot collection timing and growth stimulant treatments across four hybrids: Populus deltoides × P. nigra (Agathe F), P. maximowiczii × P. trichocarpa (Arges), P. deltoides × P. trichocarpa (Donk), and an interspecific hybrid Populus × canadensis (F-448). The experiment used hybrid poplar cuttings collected in spring 2022 and 2023, planted in controlled climates with a randomized block design. Cuttings were soaked for 24 h in growth stimulants, namely indole-3-butyric acid, cinnamic acid, and indole-3-acetic acid. After 12 weeks, rooting percentage and seedling height were assessed. The study found that the optimal time for collecting poplar cuttings for best rooting is late winter to early spring, specifically from March to early April, with shoots collected after early April showing the lowest rooting potential. The growth stimulants significantly influenced the growth of poplar seedlings. There was a tendency for lower concentrations to increase root formation and seedling height, while higher concentrations had adverse effects. Despite variations in growth rates, a consistent growth pattern was observed across different shoot collection dates for all genomic groups.

1. Introduction

Poplar (Populus spp.), belonging to the Salicaceae family, is an essential economic tree worldwide, mainly distributed in the northern temperate zone [1,2,3]. It is valued for its adaptability, fast growth, and versatile wood. Extensively cultivated in short-rotation plantations, poplar meets the rising global demand for forest products and biomass energy. Conventional and molecular breeding programs have developed superior poplar genotypes, necessitating efficient reproduction methods. Clonal propagation allows for rapid genetic improvement and quicker returns on investment [3]. Poplar hybrids offer advantages over pure poplar species by combining fast growth and high survival rates, better adapting to local climatic conditions, and improving stem quality. These benefits make hybrids superior for maximizing productivity and product value in tree breeding programs, particularly in regions with challenging climates [4]. Poplar species are propagated through stem cuttings, with the development of adventitious roots being crucial for preserving genetic traits and achieving superior cultivar production [5].

Root regeneration in cuttings is influenced by plant growth regulators, stock plant age, cutting time, and shoot position [6]. Adventitious roots typically develop from injured or hormonally stimulated stems, leaves, or other organs. The formation process shares common steps and mechanisms with lateral root formation [7].

Research on genetic variations in the rooting ability of Populus cuttings has shown that this trait is genetically controlled and significantly influenced by the poplar genotype [8,9]. Key factors include origin, clone-origin interaction, and planting date interaction. Selecting genotypes with higher rooting ability is essential to avoid poor survival rates and economic losses [9], as low rooting ability often limits cloning efforts [10].

Seasonal fluctuations in carbohydrates and hormones greatly influence the regeneration of cuttings, so it is essential to utilize the optimal period of physiological dormancy to induce adventitious rooting in hardwood cuttings successfully [8,11]. Summer is optimal for rooting softwood cuttings when donor plants are in prime physiological condition [12]. Frey et al. [11] found that the age of stock plants influences cuttings’ rooting ability, with older plants having lower potential due to increased production of rooting inhibitors as the plant lignifies. Soil and air temperature, soil moisture, and density play significant roles in root initiation and growth [13]. Pretreatment, including the intensity of bud removal, affects the rooting process, with higher bud removal intensity inversely affecting root development [14]. Several physiologically active substances, such as auxins, cytokinins, gibberellins, and ethylene, are examples of those that positively influence rooting and stimulate growth. More specifically, auxin is critical for patterning, morphogenesis, cell elongation, division, and root apical meristems. Its biosynthesis, transport, and signaling are vital for root growth and development [15,16,17]. However, high concentrations or prolonged exposure to the auxin inhibit root growth and lead to the death of cuttings. The main auxins in poplar cuttings are indole acetic acid (IAA) and indole butyric acid (IBA) [18]. IAA induces root primordium formation by causing horizontal cell splits, promoting root formation [19]. IBA is more effective than IAA in inducing rooting and promoting root primordia formation with minimal impact on subsequent root development [12].

Cytokinins, synthesized in the root system and transferred via the xylem, are crucial for growth and development, including shoot and root meristem formation and seed germination [20]. Their influence depends on concentration and the root initiation stage [21]. Cytokinins antagonize auxins during root development by inhibiting root elongation and branching, disrupting lateral root formation, and delaying cell division [22].

Gibberellins influence growth by inhibiting initial cell divisions during rooting but inducing rooting in stem cuttings from etiolated plants and stimulating adventitious shoot elongation [23,24]. Ethylene regulates root meristem differentiation, causing cell division in the quiescent center and inhibiting the differentiation of peripheral primitive cells. An increase in ethylene levels early in the poplar rooting process may be the initial biochemical event associated with rooting [25]. Cinnamic acid, a naturally occurring compound in many plants, is known to stimulate root formation, accelerate the appearance of calluses and roots, increase their quantity, and enhance the growth of aerial parts. Overall, hormones do not act independently in influencing rooting ability. The physiological state of the cutting at collection affects hormone levels, while others are influenced by the rooting medium or plant genotype. Therefore, genetic studies support the role of plant growth regulators in root development [8,9]. However, the interaction between endogenous and exogenous factors complicates establishing general rules in this complex area, indicating the need for more extensive research [12].

This study investigated the rooting and growth of poplar cuttings, focusing on the timing of shoot collection and treatment with three growth stimulants: IBA (indole butyric acid), cinnamic acid, and IAA (heteroauxin).

2. Materials and Methods

2.1. Plant Material and Study Design

The experiment was conducted in the greenhouses at the Lithuanian Research Centre for Agriculture and Forestry and in the nurseries of the Dubrava and Panevėžys divisions of the State Forest Enterprise during the vegetation seasons in 2022 and 2023. Cuttings were taken from hybrid poplars growing in the plantations at the Dubrava division in the Spring of 2022 and 2023. The study design included two parallel experiments: the first, evaluating the timing effect of shoot collection and the clone effect and the second, evaluating growth stimulants and clone effects on poplar cutting rooting and height growth.

In the first experiment, aiming to evaluate the timing effect of shoot collection and clone effect, four poplar genomic groups (hybrids) were examined: P. deltoides × P. nigra (Agathe F); P. maximowiczii × P. trichocarpa (Arges); P. deltoides × P. trichocarpa (Donk), and an interspecific hybrid P. × canadensis (F-448). Cuttings for planting in experiments were cut from March 1 until the leaves began to bloom, once every ten days (a total of five times). The cuttings were collected from the branches in the tree crown’s upper part. For this purpose, vigorously growing trees (8–10 years) with 10 cm in DBH (1.3 m above ground level) and 8–11 m in height were selected. The shoots of 50–60 cm in length were cut off with secateurs and wrapped in moist cotton to avoid desiccation during transportation from the collection site to the propagation site. Cuttings 10–12 cm in length and 1–2 cm in diameter were prepared immediately after shoot collection, i.e., on collection day. The cuttings with at least one primary bud not more than 2 cm from the top of each cutting were prepared. The prepared cuttings were sealed in polyethylene bags and stored at 2 °C. The harvested material was stored in cold storage for forest plants until planting. Thirty-five cuttings were prepared for each test variant from one poplar hybrid. Unrooted vegetative cuttings were planted in one cutting per growing container at the end of April. Treatments were randomly assigned to 275 cm⁻³ Plantek 35F containers (The BCC growing systems, multi-cell growing trays), filled with JSC “Durpeta” peat substrate for professional growers, fertilized with 3.5 g/L slow-release fertilizer (Osmocote, 17:6:10, plus minor elements) mixed throughout the potting medium.

The study was conducted under controlled climate conditions in a randomized block design with three replications, each consisting of 35 cuttings. Three containers containing 35 poplar cuttings were planted for each test variant, resulting in 105 cuttings per poplar hybrid. Specifically, each container with 35 cuttings represented a single replicate, and the three containers represented three replicates. All containers were placed on a greenhouse bench under similar conditions for the 12 weeks of the experiment. They were placed in a growth chamber with temperature settings of 22 °C during the day and 20 °C at night, humidity levels of 85% during the day and 60% at night, and a 16 h light period each day. They were sub-irrigated continuously over the remainder of the experiment.

In the second experiment, aiming to evaluate growth stimulants and clone effects, the cuttings of four poplar genomic groups (hybrids) (P. deltoides × P. nigra, P. maximowiczii × P. trichocarpa, P. deltoides × P. trichocarpa, and an interspecific hybrid P. × canadensis) were collected in the second half of March and planted at the end of April. Before planting, the cuttings were soaked in aqueous solutions of physiologically active substances (growth stimulants) for 24 h. Untreated cuttings used as controls were soaked in water. After 24 h of soaking, the cuttings were immediately inserted in rooting trays and exposed to growth chamber conditions. A 24 h soaking period helped restore lost moisture, bringing all cuttings to a consistent condition before planting. Cuttings stored in the refrigerator for a longer time lose more moisture than those prepared later and stored for a shorter time. Although the manufacturers of the growth stimulators used in this study recommended a soaking time of 16–20 h, we chose a soaking period of 24 h to equalize the physiological status of all cuttings.

For the experiment, three growth stimulants were used: indole-3-butyric acid, 1H-indole-3-butanoic acid, IBA (Kornevin^®, “Gardener’s Green Pharmacy” Ltd., Minsk, Belarus) with a concentration of 0.002%; cinnamic acid (Cirkon^®, JSC “NEST—M”, Minsk, Belarus) at concentrations of 0.0001% and 0.005%; and heteroauxin, 98%TC IAA Indole-3-Acetic Acid, IAA (Shijiazhuang Ageruo Biotech Co. Ltd., Shijiazhuang, China) at concentrations of 0.02% and 0.2%.

Clones were arranged in randomized complete blocks to minimize the effects of any potential environmental gradients in the greenhouse. The entire length of the cutting was planted in the substrate using Plantek 35F growing containers filled with JSC “Durpeta” peat substrates for professional growers. The cuttings were grown for 12 weeks in a greenhouse under natural and artificial lighting (450 mmol m⁻² s⁻¹ photons photosynthetically active radiation (PAR) at pot level to provide a 16 h photoperiod). Day temperatures were adjusted to 21 °C and night to 18 °C. The cuttings were fertilized on days 14, 28, and 42 with a solution of 28N-14P-14K mixed at a concentration of 200 ppm.

2.2. Measurements

At the end of each experiment, which coincided with the vegetation season, the cuttings were assessed for rooting (%) and seedling height (cm) according to the methodologies given in the Regulations for Reforestation and Afforestation in Lithuania [26]. These are basic methods commonly used to evaluate the growth of tree seedlings in forest plantations. The rooting of the cuttings was assessed indirectly by observing shoot growth, with an evaluation conducted at the end of the vegetation season. A seedling was considered rooted if the above-ground part of the seedling appeared healthy and vigorous. Direct assessment of the root system was not performed due to the constraints of container growth, where limited space restricts root development. The containers used tend to shape the root system such that once the roots reach the container walls or cracks, they cease to grow, with the root tips effectively pruned by air exposure. As mentioned above, balanced fertilization and watering were applied throughout the experiment to ensure uniform conditions. The seedling height/shoot length was measured from the point of the emergence on the cutting to the shoot tip.

2.3. Statistical Analysis

All data were evaluated as a two-way factorial (treatment  × hybrid) with ANOVA using PROC GLM in SAS software (SAS Institute Inc., 2002–2012, version 9.4, Cary, NC, USA) [27]. ANOVA tests for significance used the F groupwise comparison test for statistical significance. The F test was used for data evaluation, which compared the variance in each group mean to the overall variance in the dependent variable. If the variance within groups is smaller than the variance between groups, the F test will find a higher F value and, therefore, a higher likelihood that the difference observed is real and not due to chance.

Whenever treatment, hybrid, or treatment × hybrid interactions were found to be significant (p < 0.05), multiple comparisons analyses (with Tukey’s adjustment) were conducted to identify statistically significant differences between the adjusted least squares means.

3. Results and Discussion

3.1. Effect of Shoot Collection Time on Seedling Rooting

The data showed differences between the hybrids, with the percentage of rooted seedlings depending on the hybrid crossing combination (genomic group) and the date of shoot collection (

Table 1. Effect of shoot collection date on the rooting of cuttings from four poplar genomic groups at the end of the experiment.

Date of Shoot Collection	Rooting of Cuttings (%)
	P. deltoides × P. nigra (Agathe-F)	P. maximowiczii × P. trichocarpa (Arges)	P. deltoides × P. trichocarpa (Donk)	P. × canadensis (F-448)
1 March	88.6 ± 3.4	81.3 ± 3.1	80.1 ± 3.1	87.4 ± 3.4
10 March	88.4 ± 3.3	85.0 ± 3.0	81.9 ± 3.0	90.6 ± 3.5
30 March	93.4 ± 3.5	86.5 ± 3.1	91.6 ± 3.1	91.9 ± 3.5
10 April	95.7 ± 3.6	83.1 ± 3.0	93.7 ± 3.2	93.0 ± 3.2
20 April	93.1 ± 3.5	80.5 ± 2.9	86.4 ± 3.0	90.0 ± 3.4

). This information is valuable for determining the optimal timing for collecting cuttings for propagation. Leafless cuttings, collected before bud burst, rooted significantly (p ≤ 0.05) better than cuttings taken at the end of April when poplar hybrids are preparing for active growth. Based on the date of shoot collection, there was broad genotypic variation for rooting.

The date of shoot collection and rooting percentage varied among the different poplar hybrids, with some showing clear trends while others indicated higher variability. Each hybrid showed a specific trend in rooting percentages over the time of shoot collection. Genomic group P. deltoides × P. nigra showed the highest rooting percentages (Table 1). Starting from March 1, rooting percentages slightly increased over time, reaching their peak on 10 April (95.7%). However, a slight decrease was observed after 10 April (93.1%). The trend suggests that later shoot collection dates may result in slightly higher rooting percentages for this hybrid.

The P. maximowiczii × P. trichocarpa genomic group demonstrated a relatively stable rooting percentage with minor variations (Table 1). The highest rooting percentage was found around 30 March (86.5%), followed by a slight decrease afterwards. No clear trend indicated a consistent improvement or decline in rooting percentage with later shoot collection dates. The hybrid P. deltoides × P. trichocarpa showed an increasing trend in rooting percentages with later shoot collection dates, peaking around 10 April (Table 1). After this date, there was a slight decrease in rooting percentage. This hybrid showed a more consistent improvement in rooting percentage as the shoot collection date was later. The hybrid P. × canadensis demonstrated a clear trend of improved rooting percentage with later shoot collection dates. The highest rooting percentage, 93%, was observed when the shoot collection date was around 10 April. There was a significant increase in rooting percentage at later dates when the shoot collection date was between March and April.

Multiple comparison analyses of the shoot collection date and genomic group factors showed significant differences, suggesting that both impact the final percentage of poplar seedling rooting. The date of shoot collection had a statistically significant effect on the seedlings rooting, as indicated by the low p-value (<0.05) and the F-statistic (10.7) exceeding the critical F-value (2.6). The genomic group factor also had a statistically significant effect on the outcome, with a very low p-value and a high F-statistic (F > F crit). The interaction between the date of shoot collection and the genomic group was statistically significant, as indicated by the low p-value (p < 0.05) and the F-statistic (2.08) exceeding the critical F-value (2.0). Overall, both factors, as well as their interaction, had a significant impact on the final percentage of poplar seedlings rooting.

According to Zalesny [8], the shoot collection time significantly impacts the rooting of poplar cuttings. Dormant cuttings collected in late winter developed more rapidly than those collected in early winter. This more rapid development should benefit the early growth and survival of seedlings in the field. Generally, the optimal time to collect poplar cuttings for rooting is during the dormant season, typically from late winter to early spring before bud break. During this period, the trees are in a state of dormancy and are not actively growing. Dormant cuttings generally have higher sugar and total carbohydrate content and peroxidase enzyme activity, which may improve rooting [28]. These parameters were found to be positively related to rooting response.

Our study showed that storing the cuttings for an extended period can negatively influence rooting success. The longer cuttings are stored, the more likely they are to dehydrate, lose viability, and become more susceptible to disease or pathogens. While storing cuttings can be a useful strategy for extending propagation seasons or transporting material over long distances, it is essential to minimize storage time and take appropriate measures to maintain cutting viability and health. However, collecting cuttings in the late season can negatively impact rooting success. Cuttings collected in the late season typically have higher nitrogen content, which may stimulate shoot development but could negatively affect rooting by competing for carbohydrates, nutrients, and hormones [29].

However, the specific timing within the dormant season can vary depending on local climate conditions. Therefore, temperature and moisture levels should be considered to determine the optimal time for shoot collection.

Our findings, in line with previous studies, showed lower rooting for cuttings collected from shoots after 10 April. During this period, shoots emerge from dormancy and allocate resources to aboveground growth instead of keeping them within the shoot [8,30].

3.2. Effect of Shoot Collection Time on Seedling Height Growth

For each hybrid, the height of the seedlings at the end of the vegetation season varied depending on the shoot collection date (

Table 2. The effect of the shoot collection date on the seedling height (cm) of the rooted cuttings of four poplar genomic groups at the end of the vegetation season.

Date of Shoot Collection	Height (cm)
	P. deltoides × P. nigra (Agathe-F)	P. maximowiczii × P. trichocarpa (Arges)	P. deltoides × P. trichocarpa (Donk)	P. × canadensis (F-448)
1 March	124.47 ± 11.71	122.21 ± 11.15	84.78 ± 12.31	115.28 ± 12.15
10 March	132.22 ± 10.20	118.63 ± 11.17	90.77 ± 16.22	120.48 ± 10.27
30 March	131.77 ± 21.11	123.81 ± 14.31	89.18 ± 11.05	123.42 ± 11.12
10 April	135.93 ± 19.09	121.91 ± 14.61	104.23 ± 21.18	118.38 ± 12.23
20 April	123.04 ± 21.24	115.28 ± 21.79	85.09 ± 15.26	113.76 ± 17.77

). An increase in height was found for those seedlings grown from shoots collected between March 1 and April 10, followed by a decrease or stabilization in height by April 20 for most hybrids. Also, the trends in height growth varied among the hybrids. While some hybrids showed relatively stable or increasing growth patterns, others showed no clear trend.

Seedlings of genomic group P. deltoides × P. nigra were the tallest (Table 2). There was an increased trend in height for those seedlings grown from shoots collected from 1 March to 10 April, followed by a slight decrease by 20 April. P. maximowiczii × P. trichocarpa seedlings showed no clear trend—no increase or decrease in height for the evaluation dates. There was an increasing trend in the height of seedlings of genomic group P. deltoides × P. trichocarpa, observed from 1 March to 10 April, followed by a slight decrease by 20 April. However, the growth rate did not vary significantly, particularly between 10 March and 30 March. Seedlings of genomic group P. × canadensis showed a relatively steady increase in height from 1 March to 30 March. This growth trend was more consistent compared to other hybrids.

The height variation may be influenced by the shoot development stage at the collection time, environmental conditions during the growth period, and each hybrid’s inherent growth characteristics [30]. Genetic differences, environmental conditions, and interactions between these factors likely contribute to the observed variations in height growth [12].

After adjusting for multiple comparisons, it was found that both the date of shoot collection and the genomic group significantly affected seedling height. At the same time, their interaction did not significantly influence height variation. The shoot collection date had a statistically significant effect on height (F-statistic 6.28) exceeding the critical F-value (2.40), p < 0.001). This suggested that there were differences in height among the different collection dates. The genomic group also had a highly significant effect on height (F (132.7) > F crit (2.64), p < 0.001), indicating significant differences in height among the different hybrid groups. There were differences in height among individual samples within the same hybrid group. The interaction between the date of shoot collection and the genomic group was not statistically significant (F (1.51) ≤ F crit (1.78), p = 0.120). Therefore, the effect of the shoot collection date on height did not vary significantly across different genomic groups. Previous studies explored the correlation between shoot collection date and the growth of poplar seedlings [5,8]. A significant relation was found between shoot collection date and subsequent seedling height, biomass accumulation, and root development. Early-season collections tended to cause greater height and biomass, suggesting an optimal shoot collection time for maximizing growth parameters.

The shoot collection date significantly affected seedling growth in height rates over the vegetation season, with the height growth rates varying for each hybrid.

The growth rate of cuttings of genomic group P. deltoides × P. nigra collected between 1 March and 30 March showed consistent growth patterns, with only slight variations in growth rates (Figure 1). They showed robust growth throughout the study period. These cuttings demonstrated higher growth rates compared to those collected later in April. The obtained findings suggested that shoot collection during the period from 1 March to 30 March is optimal for promoting healthy and vigorous growth in poplar cuttings for P. deltoides × P. nigra genomic group. Poplar cuttings collected in late April (20 April) showed slower growth rates than earlier collection dates. This showed that delayed shoot collection may negatively affect seedling growth dynamics.

Seedlings from the hybrid P. maximowiczii × P. trichocarpa increased height from 10 May to 10 August, with rapid growth observed particularly from 10 May to 20 June (Figure 2). Towards the end of the study period (from 30 July to 10 August), there were signs of growth stabilization, characterized by a reduced rate of height increase. This may indicate that the seedlings were nearing maturity, restricting further growth. Cuttings collected earlier may stabilize sooner, whereas those from later collections might continue to show prolonged growth. Variability in growth rates was evident for different shoot collection dates. Seedlings from shoots collected on 1 March and 10 March showed slightly faster initial growth than those collected later in March or April. Despite the variations in growth rates, there was a consistent growth pattern across different shoot collection dates. This indicated that although initial growth may vary, the overall trend remained similar for all batches of cuttings.

The height dynamics of the hybrid P. deltoides × P. trichocarpa showed variation in the height of the poplar cuttings depending on when the shoots were collected (Figure 3). Cuttings collected later in the spring (beginning of April) tended to cause slightly higher height growth than those collected earlier (in March). Despite variations in the initial growth rates, seedlings from all collection dates showed a consistent growth pattern over time. Cuttings collected earlier in the spring grew more slowly at the beginning and showed more intensive growth later, while cuttings collected later grew faster at the beginning but more slowly at the end of the season. The peak of the growth depended on the shoot collection date, i.e., cuttings collected earlier reached their peak growth earlier in the season than those collected later (Figure 3). The timing of physiological processes influencing growth can explain these differences. The obtained data showed that all seedlings continued to increase in height during the vegetation season—summer months—regardless of the initial collection date.

The growth rate of genomic group P. × canadensis varied slightly depending on the shoot collection date (Figure 4). Shoots collected on 1 March had lower initial height growth than those collected later. Initial height growth was relatively low, but there was consistent growth, with noticeable increases in height at each measurement interval. Cuttings from shoots collected on 30 March had the highest growth rates throughout the study period. The growth rates of cuttings collected on 20 April were slower initially but accelerated over time, with a significant increase in height in the later measurement intervals. Despite some variations, a general trend of poplar cutting height increase occurred throughout the study period.

Summing up the trends described above, although initial height and growth rates differed depending on the shoot collection date, all cuttings showed a similar pattern of constant height increase over the study period.

3.3. Growth Stimulants and Clone Effect on Rooting

The aqueous solutions of growth regulators affected the root system formation of poplar seedlings collected from shoots of different genomic groups, with varying impacts depending on the hybrid and growth stimulant used (

Table 3. Effect of plant growth regulators and their concentration on the rooting of the cuttings of a genomic group.

Treatment, Concentration (%)	Rooting of Cuttings (%)
	P. deltoides × P. nigra (Agathe-F)	P. maximowiczii × P. trichocarpa (Arges)	P. deltoides × P. trichocarpa (Donk)	P. × canadensis (F-448)
Control	88.2 ± 3.3	82.7 ± 3.9	89.6 ± 3.6	80.6 ± 5.3
IBA *, 0.002	97.7 ± 1.9	90.1 ± 2.3	95.9 ± 1.9	88.8 ± 4.7
IAA **, 0.02	92.5 ± 2.4	87.7 ± 3.6	90.8 ± 1.4	91.2 ± 1.3
IAA **, 0.2	84.2 ± 5.6	82.2 ± 2.4	87.4 ± 4.7	82.4 ± 5.7
Cinnamic acid, 0.0001	100.0 ± 0.0	88.3 ± 4.6	93.1 ± 2.6	87.4 ± 3.6
Cinnamic acid, 0.005	91.6 ± 2.8	84.3 ± 4.8	89.6 ± 2.5	81.2 ± 4.3

Notes: * IBA is indole-3-butyric acid; ** IAA is heteroauxin, indole-3-acetic acid.

). The hybrids’ root formation differences were observed after treatment with different growth stimulants.

The highest rooting percentage was determined for the genomic group P. deltoides × P. nigra (Table 3). When affected by low concentrations of cinnamic acid (0.0001% solutions), this hybrid has 100% rooting. Also, a high rooting percentage was found under the influence of IBA 0.002% solutions. The cuttings affected by heteroauxin (0.02%) rooted better than the control—when they were soaked in water without treatment with growth stimulants. Furthermore, high concentrations of growth stimulants negatively affected the rooting of cuttings, with rooting percentages almost 10% lower than those treated with low concentrations. The effects of growth stimulants were also different for different genomic groups (crossing combinations). The genomic groups P. maximowiczii × P. trichocarpa and P. deltoides × P. trichocarpa showed the best rooting results after the 0.002% IBA stimulator treatments. Meanwhile, the interspecific hybrid of P. × canadensis showed the best rooting after the stimulation with heteroauxin.

After adjusting for multiple comparisons analysis, significant differences were found in the median rooting percentage in different growth stimulants and genomic groups. Growth stimulants and the genomic group had a significant effect on the percentage of rooting (F (3.17) > F crit (2.86), p ≤ 0.05). However, the interaction between these two factors had no significant effect on the rooting of cuttings (F (2.11) < F crit (2.25), p > 0.05).

Several theories explain propagation through stem cuttings in woody plants to capture specific genetic combinations (phenotypes) and provide superior cultivars for planting. The production of nursery seedlings should ensure high rooting of cuttings; otherwise, the cost of growing seedlings will increase. Root growth in cuttings is affected by various plant growth regulators and stimulants, including those used in our study. p-Hydroxy benzoic acid (p-HBA), paracoumaric acid, and ferulic acid have been reported to enhance rooting in easy-, difficult- and obstinate-to-root species [31]. Haissig [32] identified co-factors such as phenolic–auxin conjugates and proposed several theories to explain the effect of phenolic compounds, including modification of IAA-oxidase activity and stimulation of auxin synthesis. Studies by many authors showed a positive impact of higher concentrations of growth stimulants on the rooting of cuttings [32,33]. However, our study identified better-rooting results with low-concentration solutions of growth stimulants, while high concentrations inhibited the rooting process [5]. Moreover, one purpose was to stimulate shoot development of rooted cuttings. Previous studies also showed that plant regulators increased plant height, leaf area, number of leaves, shoot mass, and secondary branches [34].

Furthermore, the use of plant regulators influenced stem height growth compared to the control group, with some treatments increasing heights while others resulted in decreased heights (

Table 4. Effect of plant growth regulators and their concentration on the plant height of the rooted cuttings of four poplar genomic groups at the end of the experiment.

Treatment, Concentration (%)	Height (cm)
	P. deltoides × P. nigra (Agathe-F)	P. maximowiczii × P. trichocarpa (Arges)	P. deltoides × P. trichocarpa (Donk)	P. × canadensis (F-448)
Control	119.47 ± 13.74	100.24 ± 13.19	85.78 ± 17.33	119.18 ± 15.15
IBA *, 0.002	138.22 ± 11.21	128.63 ± 12.18	94.77 ± 19.62	125.41 ± 14.77
IAA **, 0.02	131.77 ± 21.11	127.87 ± 18.30	89.08 ± 14.55	133.47 ± 21.12
IAA **, 0.2	112.36 ± 15.22	112.35 ± 18.28	85.55 ± 14.99	99.74 ± 19.05
Cinnamic acid, 0.0001	134.93 ± 9.59	121.91 ± 14.61	105.13 ± 20.13	127.33 ± 17.33
Cinnamic acid, 0.005	111.14 ± 20.44	115.27 ± 14.77	86.19 ± 16.56	113.96 ± 14.78

Notes: * IBA is indole-3-butyric acid; ** IAA is indole-3-acetic acid, heteroauxin.

).

For the genomic group P. deltoides × P. nigra, the increase in height was mainly affected by IBA 0.002% and slightly less affected by cinnamic acid 0.0001% (Table 4). Relatively good results were achieved when heteroauxin 0.02% was used. The hybrid P. deltoides × P. nigra responded negatively to high concentrations of growth regulators, experiencing intensified stress that minimized shoot development (height growth). Even the control group had a larger height than those treated with high concentrations of growth regulator solutions. Similar results were observed for the hybrid P. maximowiczii × P. trichocarpa, with the best growth recorded under the influence of 0.002% IBA and 0.02% heteroauxin. However, this hybrid was less sensitive to high concentrations of growth stimulants. Although the growth was reduced under the influence of high concentrations, it was higher than the control seedlings. The growth in height of P. deltoides × P. trichocarpa was predominantly stimulated by 0.0001% cinnamic acid, while 0.002% IBA was less effective. Heteroauxin showed no significant effect on the height of the hybrid, and high concentrations of growth regulators did not significantly affect its height growth. The interspecific hybrid P. × canadensis showed the highest responsiveness to the influence of 0.02% heteroauxin. However, a high concentration (0.2%) of this growth regulator had a notably strong negative effect on height growth. The treatments with IBA 0.002% and cinnamic acid 0.0001% responded with a similar positive impact on the height growth of this hybrid.

Multiple comparison analyses of growth stimulants and genomic group factors showed significant differences, suggesting that both impact poplar seedlings’ growth. The F-value for the growth stimulants factor is F (6.55) > F crit (3.46), with a p-value of p ≤ 0.05, indicating a statistically significant difference among the growth stimulants. For the crossing combinations factor, the F-value is F (9.96) > F crit (2.57) with a small p-value (p ≤ 0.05), suggesting a highly significant difference among the crossing combinations. The interaction effect of growth stimulants and the crossing combinations was not statistically significant. Therefore, the impact of crossing combinations did not depend on the levels of growth stimulants.

Hormone treatments were of varying efficacy in different genomic groups of poplar. Lower concentrations generally increased height relative to the control group, while higher concentrations sometimes decreased. Previous studies showed that phytohormones regulate essential physiological and developmental processes during a plant’s life cycle [35,36]. Research by Yuan et al. (2019) [37] also demonstrated that the growth response of Populus hybrids to biostimulants varied significantly among different genotypes, with some showing enhanced growth while others showed little to no response. Previous studies showed that IAA is important in cambial activity and wood formation of woody plants. Since then, numerous experiments using exogenous auxin treatments of both hardwoods and conifers have demonstrated the potential of IAA to affect most aspects of cambial growth in a dose-dependent manner, including xylem and phloem production and size, as well as the secondary wall thickness of xylem elements [38]. Summarizing the obtained results, the selection of growth stimulators and genomic groups significantly influenced the growth of poplar seedlings. However, the interaction between growth stimulators and genomic groups had no effect.

4. Conclusions

The current study aimed to evaluate the effect of shoot collection time and treatment with growth stimulants (IBA, indole-3-butyric acid, 0.002%; cinnamic acid, 0.0001% and 0.005%; and IAA, heteroauxin, 0.02% and 0.2%) on the rooting and height growth of cuttings collected from four poplar (Populus spp.) genomic groups (hybrids)—P. deltoides × P. nigra (Agathe F); P. maximowiczii × P. trichocarpa (Arges); P. deltoides × P. trichocarpa (Donk), and P. × canadensis (F-448). The study results showed that the shoot collection date significantly affected the rooting of the seedlings. The optimal time to collect poplar cuttings for the best rooting was found to be in late winter to early spring before bud break. Cuttings prepared from shoots collected after the first decade of April showed the lowest rooting potential. However, the specific timing within the dormant season can vary depending on the local climate conditions. The optimal time for shoot collection to maximize growth potential in poplar cuttings is from March to early April. Despite the variations in growth rates, there was a consistent growth pattern across different shoot collection dates for all genomic groups. While some hybrids showed relatively stable or increasing growth patterns, others showed no clear trend.

Aqueous solutions of growth stimulants significantly influenced poplar seedling growth. Hormone treatments showed varying degrees of effectiveness across different poplar genomic groups. Lower concentrations influenced the formation of the new root system. Generally, they resulted in increased heights compared to the control, while higher concentrations negatively affected the rooting of cuttings and sometimes resulted in decreased heights.