Effect of Plant Growth Regulators on Sweetpotato Stem Tissue Development and Structure

Abstract

Sweetpotato (Ipomoea batatas) is an important global food crop, yet propagation through greenhouse-produced slips is limited by low transplant establishment rates. Previous studies have focused on external morphological traits to improve transplant quality, but the internal anatomical structure of sweetpotato slips remains largely unexplored. This study examined the effects of four plant growth regulators (PGRs)—flurprimidol, paclobutrazol, uniconazole, and indole-3-butyric acid (IBA)—applied foliarly at varying rates to sweetpotato slips grown in a greenhouse. Cross-sections of the stem were stained with toluidine blue O and analyzed microscopically to assess epidermal, collenchyma, parenchyma, and xylem tissue thickness. Flurprimidol at 120 mg·L⁻¹ significantly increased epidermal thickness by 31.8% compared to the control. Paclobutrazol at 30 and 60 mg·L⁻¹ significantly reduced collenchyma thickness by 37.8% and 39.7%, respectively. Other treatments showed no statistically significant differences across measured tissues, although some trends were observed. These findings suggest that certain PGRs may influence internal slip anatomy, particularly the epidermis, which could improve transplant resilience and field performance. Further research is needed to optimize application rates and evaluate long-term agronomic outcomes of anatomical modifications in sweetpotato slips.

1. Introduction

Sweetpotato, Ipomoea batatas, is cultivated globally for its storage roots, ranking as the world’s sixth most significant food crop [1]. In 2024, the United States produced 1.235 million tons of sweetpotato, valued at USD 615 million [2]. While this crop plays a crucial role in food security, its production cycle is complex and often disrupted by various challenges that negatively impact yields. One notable concern is slip propagation, which involves unrooted stem cuttings from the sweetpotato plant. These slips can be generated from plant beds using storage roots from the previous year’s harvest or cultivated in greenhouses. However, greenhouse-produced slips face significant challenges, exhibiting only a 50% establishment rate when transplanted into commercial fields (Harvey, personal communication, 2023). This low survival rate is thought to stem from the protective environment of the greenhouse, resulting in slips that develop thin cell walls and become susceptible to the harsh conditions of the field, ultimately leading to their decline (Harvey, personal communication, 2023).

Research indicates that in sweetpotatoes, slips with a larger diameter contribute to higher transplant survival rates [3]. However, slip uniformity is rarely achieved, and producers do not have the time or resources to sort through and discard plant material. Additionally, previous research has explored external stem characteristics and hardening techniques to improve transplant resilience, but has no real significant effect. Relatively little is known about the internal anatomy of sweetpotato slips and its influence on transplant success [4].

The internal structure of a stem plays a crucial role in water and nutrient transport, mechanical support, and root initiation. The shoot system supports the plant’s vegetative and reproductive organs, stores resources, and connects the roots and aerial tissues [5]. Plant stems comprise three cell types: parenchyma, collenchyma, and sclerenchyma. Each contributes uniquely to the stem structure, with parenchyma supporting storage, metabolism, and regenerative growth, collenchyma providing flexible support in young tissues, and sclerenchyma offering rigid structural reinforcement in mature stems via lignified secondary walls [5]. The various tissues also play critical roles in stem function. The epidermis, a component of the dermal system, acts as a barrier against environmental stress, minimizes water loss through a waxy cuticle, regulates gas exchange via stomata, and contributes to structural integrity [6,7]. The vascular system, composed of xylem for the upward transport of water and minerals and phloem for distributing photosynthates, enhances stem strength through lignified structural elements such as tracheids and vessel elements [8,9].

These structural tissues are functionally important, and their development is regulated by hormonal signaling and genetic pathways originating from stem cell activity within the shoot apical meristem [10]. Plant hormones such as auxin, gibberellin, and cytokinin manage the spatial organization of stem tissues—such as the epidermis, cortex, and vascular bundles—highlighting the complexity of stem architecture during vegetative growth. Recent molecular research further supports that stem development is not a product of tissue differentiation but is governed by hormone–gene networks. According to Peng et al., these phytohormones coordinate with transcription factors and non-coding regulatory RNAs to control both stem elongation and thickening in vascular plants, ultimately influencing their structure and resilience to environmental stresses [11].

At the cellular level, auxin and cytokinin also play essential roles in cell differentiation. A study found during somatic embryogenesis, a high auxin-to-cytokinin ratio induces callus formation, followed by cytokinin activating key genes such as WOX5 and PLETHORA, which promote organized tissue development [12]. Although these mechanisms are best characterized in embryogenic systems, they suggest that exogenous manipulation of hormone levels—even in non-embryonic tissues—can influence internal structure by altering developmental signals.

Plant growth regulators are natural or synthetic compounds that influence physiological and developmental processes in plants. Despite their broad use in horticulture, PGRs have rarely been investigated in sweetpotato production. Previous studies focused primarily on yield effects or height control in vitro [13]. However, the internal structural changes that may result from PGR applications on sweetpotatoes remain largely unexplored. In contrast, studies in other crops have demonstrated that PGR application can affect internal stem anatomy. For example, uniconazole application in Oryza sativa increased culm diameter by 10% and parenchyma cell wall thickness by up to 65.2% [14]. Similarly, paclobutrazol applied at varying concentrations affected stem, leaf, and tracheal diameter in chrysanthemums, with higher rates promoting greater structural thickening [15]. Flurprimidol application also resulted in measurable increases in epidermal cell size in Geogenanthus undatus [16]. In Zanthoxylum beecheyanum, IBA promoted cortical cell activity and cambial development during the rooting induction phase, highlighting its role in cellular differentiation [17].

Foliar applications of PGRs such as paclobutrazol and uniconazole have also been shown to alter stem architecture—reducing elongation and modifying branching patterns in multiple Kalanchoe species [18]. These structural changes are often controlled by shifts in internal anatomy, suggesting that PGRs can influence tissue differentiation and cellular organization. These findings underscore the potential for PGRs to induce significant anatomical modifications in plants, warranting further investigation into their structural effects in sweetpotato.

In the current investigation, four PGRs—flurprimidol, paclobutrazol, uniconazole, and IBA—were applied foliarly at varying rates to 14-day-old sweetpotato slips. The slips were harvested 7 days post-application, and microscopic analysis was conducted on stained stem cross-sections to examine the thickness of various stem structures. This study aims to assess how different PGRs and application rates influence the internal stem structure in sweetpotato slips, focusing on the thickness of major tissue types.

2. Materials and Methods

2.1. Plant Growth Conditions

The trial was conducted in a single greenhouse bay at Mississippi State University, Starkville, MS, USA, and the greenhouse maintained a ventilation setpoint of 78 degrees Fahrenheit, average humidity of 70–80%, and under a natural photoperiod from 19 February to 15 March 2023. Virus-tested, two-node ‘Beauregard’ (B-14) sweetpotato slips were transplanted into 38-cell trays filled with a soilless substrate. Regular monitoring of moisture content and plant health ensured the slips were adequately watered and received a single application of 20-8.8-16.6 fertilizer at 200 ppm (Peters Professional 20-20-20) (Everris NA Inc., Dublin, OH, USA).

2.2. Treatments and Application

Four PGRs were evaluated as follows: three anti-gibberellins—flurprimidol (TopFlor^®, 0.38% a.i.; SePRO Corp., Carmel, IN, USA), paclobutrazol (Piccolo 10 XC^®, 4.0% a.i.), and uniconazole (Concise^®, 0.055% a.i.)—and one auxin, indole-3-butyric acid (IBA; Advocate^®, 20% a.i.; Fine Americas Inc., Walnut Creek, CA, USA). Stock solutions were diluted with distilled water to achieve final concentrations of 20, 60, and 120 mg·L⁻¹ (flurprimidol); 30, 60, and 120 mg·L⁻¹ (paclobutrazol); 10, 20, and 30 mg·L⁻¹ (uniconazole); and 250, 500, and 750 mg·L⁻¹ (IBA). Target concentrations were selected based on manufacturer recommendations and preliminary studies. Each solution was prepared using a calibrated pipette and applied as foliar sprays at 30 mL per tray using a hand-held sprayer.

2.3. Experimental Design and Outline

A complete randomized design was used to evaluate the effects of PGR on sweetpotato slips. The experiment included 13 38-cell trays, with each tray assigned to 1 of 12 PGR treatments or a water-treated control. Each tray contained 15 slips, which were randomized on a greenhouse bench to ensure uniform environmental exposure.

The trial lasted 28 days. On 19 February 2024, slips were transplanted and randomized within the tray. From 26 February to 4 March (days 7–14), fertilizer was applied to promote root establishment. Foliar application of PGRs occurred on 5 March (day 15). On 11 March (day 22), five slips were randomly selected from each tray as representative samples for anatomical evaluation, conducted between 11 and 16 March (days 22–27).

2.4. Observational Data and Staining Evaluation

On day 22, five representative slips were harvested per tray using hand pruners for anatomical evaluation. The primary stem of each slip was divided into three segments—base, middle, and tip—and three complete cross-sections were obtained from each segment using a straight razor blade. Cross-sections were stained with toluidine blue O (TBO) following the free-hand sectioning and staining protocol described in Chapter 9 of Yeung’s book, “A Beginner’s Guide to the Study of Plant Structure” [19].

Stained sections were observed under a compound microscope and analyzed using Motic Images Plus 3.1 software (Motic, Kowloon, Hong Kong). Four stem tissues—epidermis, collenchyma, parenchyma, and xylem—were measured to evaluate their contribution to stem structure and stability. Five evenly spaced measurements were taken around the circumference for each tissue and averaged to determine the mean width (Figure 1).

2.5. Statistical Analysis

Data were analyzed using a mixed-effects model in SAS 9.4 (SAS Institute, Cary, NC, USA), accounting for the nested structure of slips within a tray, which were treated as random effects to account for variation across experimental units. Treatment (PGR) was considered a fixed effect for all cross-sectional metrics. The Satterthwaite approximation was used to estimate degrees of freedom. Each stem tissue measurement was analyzed with respect to treatment, with results reported as least squares means or covariate-adjusted least squares means, depending on model specification. Statistical significance was set at p ≤ 0.05. Each PGR was analyzed independently for each tissue structure and compared to the control, resulting in four different PGR vs. control analysis. The use of a mixed-effects model was justified by the inclusion of tray-level variability and the hierarchical design of the experiment, which required accounting for potential intra-tray correlation to avoid inflated Type I error rates.

While increased tissue thickness was hypothesized to benefit propagation, a two-sided test framework was chosen to remain conservative and allow for detection of both increases and decreases in tissue structure metrics. All hypothesis tests were conducted as two-sided comparisons unless otherwise stated. It is important to note that the sample size was limited due to logistical constraints, which may have reduced the statistical power to detect subtle anatomical differences.

3. Results

Epidermal thickness in sweetpotato slip cross-sections varied in response to chemical treatments and application rates. Mean values ranged from 2.12 µm in the uniconazole treatment at 30 mg·L⁻¹ (U-30) to 3.07 µm in the flurprimidol treatment at 120 mg·L⁻¹ (F-120). Standard deviations ranged from 0.15 to 0.27 µm, reflecting moderate treatment variability. Most treatments did not result in significant changes in epidermal thickness and were comparable to the control. The exception was flurprimidol at 120 mg·L⁻¹ (F-120), which significantly increased epidermal thickness by 31.8% compared to the control (p = 0.029). These findings suggest that while most PGR applications had a limited impact on epidermal development, flurprimidol at higher concentrations may enhance structural growth in sweetpotato slip epidermal tissue (

Table 1. Sweetpotato slip structural property mean and standard error for treatments, flurprimidol, paclobutrazol, indole-3-butyric acid, and uniconazole. Structures measured include collenchyma, epidermis, parenchyma, and xylem. Differences in treatments are denoted by different letters. p-values are rounded to three decimal places; significant differences (p < 0.05) are indicated with an asterisk (*).

Flurprimidol
Stem Structure	Control	F20	F60	F120	p Value
Collenchyma	9.04 ± 1.8	6.31 ± 1.8	8.31 ± 1.8	9.45 ± 1.8	0.611
Epidermis	2.33 ± 0.2 (b)	2.77 ± 0.2 (ab)	2.30 ± 0.2 (b)	3.07 ± 0.2 (a)	0.029 *
Parenchyma	1.59 ± 0.3	1.67 ± 0.3	1.30 ± 0.3	2.30 ± 0.5	0.467
Xylem	5.92 ± 0.8	5.51 ± 0.8	4.52 ± 0.6	5.59 ± 0.6	0.514
Paclobutrazol
Stem Structure	Control	P30	P60	P120	p Value
Collenchyma	9.04 ± 1.0 (a)	5.62 ± 1.0 (b)	5.45 ± 1.0 (b)	9.34 ± 1.0 (a)	0.049 *
Epidermis	2.33 ± 0.3	2.36 ± 0.3	2.43 ± 0.3	2.75 ± 0.3	0.691
Parenchyma	1.59 ± 0.2	1.71 ± 0.2	1.30 ± 0.1	1.44 ± 0.1	0.407
Xylem	5.92 ± 1.0	6.62 ± 1.0	4.50 ± 0.8	4.53 ± 0.8	0.328
Indole-3-butyric acid
Stem Structure	Control	IBA 250	IBA 500	IBA 750	p Value
Collenchyma	9.04 ± 3.4	8.91 ± 3.4	12.63 ± 3.4	9.56 ± 3.4	0.850
Epidermis	2.33 ± 0.2	2.71 ± 0.2	2.81 ± 0.2	2.79 ± 0.2	0.507
Parenchyma	1.59 ± 0.2	1.36 ± 0.2	1.69 ± 0.3	1.46 ± 0.2	0.677
Xylem	5.92 ± 0.7	4.67 ± 0.7	6.07 ± 0.7	4.53 ± 0.6	0.352
Uniconazole
Stem Structure	Control	U10	U20	U30	p Value
Collenchyma	9.04 ± 2.4	8.78 ± 2.4	7.86 ± 2.4	7.18 ± 2.4	0.938
Epidermis	2.33 ± 0.2	2.73 ± 0.2	2.54 ± 0.2	2.12 ± 0.2	0.094
Parenchyma	1.59 ± 0.2	1.05 ± 0.2	1.21 ± 0.2	1.30 ± 0.2	0.386
Xylem	5.92 ± 0.7	5.45 ± 0.6	4.34 ± 0.6	4.52 ± 0.6	0.313

).

Analysis of parenchyma thickness revealed minor variations among treatments, though none were statistically significant when compared to the control. Flurprimidol had the largest average thickness, with the 120 mg·L⁻¹ treatments resulting in a thickness of 2.30 µm. Paclobutrazol and IBA treatments produced intermediate parenchyma thickness values, ranging from 1.71 µm at 30 mg·L⁻¹ (P-30) to 1.30 µm at 60 mg·L⁻¹ (P-60), and from 1.69 µm at 500 mg·L⁻¹ (IBA-500) to 1.36 µm at 250 mg·L⁻¹ (IBA-250). Interestingly, uniconazole treatments resulted in the thinnest parenchyma measurements overall, with the 30 mg·L⁻¹ rate producing the greatest thickness among them at only 1.30 µm. The lowest parenchyma thickness was observed in the 10 mg·L⁻¹ treatment, measuring 1.05 µm. These results suggest that higher concentrations do not result in increased parenchyma thickness, highlighting potential complexity in PGR interactions with stem tissue development (Table 1).

Analysis of collenchyma thickness showed no significant differences among the majority of treatments compared to the control (9.04 µm). Flurprimidol treatments increased in average thickness when the concentrations increased, from 6.31 µm at 20 mg·L⁻¹ to 8.31 µm at 60 mg·L⁻¹, and 9.45 µm at 120 mg·L⁻¹. In contrast, uniconazole treatments showed a decreasing trend, with the lowest concentration of 10 mg·L⁻¹ averaging 8.78 µm and the highest, 30 mg·L⁻¹, producing the thinnest value at 7.18 µm. IBA treatments did not follow a distinct trend; the 500 mg·L⁻¹ rate resulted in the thickest collenchyma (12.63 µm), while the 250 and 750 mg·L⁻¹ rates averaged 8.91 µm and 9.56 µm, respectively. Similarly, paclobutrazol treatments showed no consistent trend but did significantly decrease the collenchyma width by 37.8% and 39.7% at the 30 and 60 mg·L⁻¹ rates, whereas the 120 mg·L⁻¹ rate was comparable to the control (p = 0.049). Overall, most treatments did not significantly increase collenchyma thickness, though reductions observed with paclobutrazol highlight the complex responses of plant tissues to growth regulators (Table 1 and Figure 2).

Analysis of xylem thickness revealed no statistically significant differences among treatments compared to the control. Additionally, there were no distinct trends among treatments or concentrations. Standard deviations ranged from 0.6 to 1.0 µm, reflecting moderate treatment variability. The largest mean values derived from the lowest paclobutrazol concentration of 30 mg·L⁻¹ (P-30) with a thickness of 6.62. Uniconazole treatment of 20 mg·L⁻¹ produced the lowest xylem thickness overall of 4.34 µm. These results suggest that while some structures exhibited observable trends, no consistent or statistically significant patterns were detected across the xylem (Table 1 and Figure 2).

4. Discussion

To evaluate the effects of four PGRs on internal stem structure, stained cross-sections of sweetpotato slips were analyzed. The results revealed a limited number of statistically significant effects. These effects, while isolated to individual PGR–tissue combinations, suggest the potential for treatment-specific structural responses. The epidermal layer exhibited the most notable treatment-related differences.

Flurprimidol at 120 mg·L⁻¹ (F-120) produced the greatest epidermal thickness, increasing by 31.8% over the control. Other flurprimidol rates also yielded greater mean thicknesses than most treatments, suggesting a stimulatory effect on epidermal development. Similar epidermal enlargement was reported in Geogenanthus undatus ‘Inca’ from 40 µm in length and 33 µm in width to 70 µm by 50 µm following flurprimidol application [16]. Although the mechanism remains uncertain, flurprimidol’s inhibition of GA₁ biosynthesis likely reduces cell elongation and alters cell expansion dynamics in epidermal tissue; thus, their suppression may lead to more compact, radially thickened epidermal cells [20]. Inhibition of gibberellin signaling can also shift hormonal balance toward cytokinin activity, which has been associated with increased cell division in epidermal layers [21,22]. These hormonal interactions may underlie the observed increase in epidermal width following flurprimidol application. A thicker epidermis may improve plant water retention, protection, and pathogen resistance. Such adaptations have been observed in xerophytic species like Aloe vera, where epidermal thickening aids in minimizing water loss under arid conditions [23].

Parenchyma thickness varied across treatments, though no clear trends or significant differences were observed. While previous studies have demonstrated parenchyma thickening in response to PGR, the current findings did not reflect such effects. For example, uniconazole increased parenchyma cell wall thickness by 43.7% and 65.2% under normal and shaded conditions, respectively, and altered cell morphology through suppression of endogenous auxin and gibberellic acid levels [14]. Gibberellins are known to regulate cell elongation and expansion in plants and their stems [24]. Suppression of these hormones by growth retardants like uniconazole may restrict cell elongation while allowing radial thickening. Paclobutrazol has also shown variable effects depending on concentration and cultivar. In Chrysanthemum, higher concentrations increased stem and tracheal diameters, while lower concentrations enhanced the size of parenchyma and adjacent tissues [15]. Although species-specific responses are well-documented, these hormonal pathways are conserved across many dicots, which may explain the limited anatomical response observed in sweetpotato slips in the present study. Future studies should prioritize dose–response experiments and evaluate cultivar-specific sensitivity to better determine the conditions under which PGRs may enhance parenchyma development in this crop.

The trend of collenchyma increasing widths corresponding with higher rates was not observed in other studies. For instance, IBA application in Carpinus betulus L. ‘Fastigiata’ did not significantly affect collenchyma thickness, which remained near 46 µm in both treated and control shoots [25]. In contrast, flurprimidol-treated Geogenanthus undatus ‘Inca’ showed an increase in collenchyma, occupying half of the cortex—a one-sixth increase over the control—though this response lacked a consistent dose-dependent pattern [16]. Studies involving paclobutrazol have reported both inhibitory and stimulatory effects: collenchyma layers were reduced with increasing concentrations in yellow passion fruit [26], whereas in Chrysanthemum ‘Jaguar Red’, the cortex thickened most at 100 ppm, with no notable changes at 50 or 150 ppm [27]. These inconsistencies highlight species- and cultivar-specific sensitivity to PGRs and do not fully explain the lack of effect observed at the highest rate and the inhibitory responses at lower concentrations in the current study.

Collenchyma thickening is driven primarily by enhanced primary wall deposition, often influenced by hormones within the plant. Auxins contribute to collenchyma development by promoting the cell wall changes necessary for expansion, and this process is regulated by auxin-responsive genes and influenced by the cellular context [28]. In contrast, growth retardants like flurprimidol and paclobutrazol inhibit gibberellin biosynthesis, which could reduce longitudinal expansion while indirectly promoting radial cell thickening [29]. The lack of a dose-dependent pattern in our study may suggest a threshold response or interaction with endogenous hormone levels specific to sweetpotato slips.

The xylem results indicated no statistically significant differences between the treatments and the control group. However, a general trend showed that most treatments exhibited a thinner xylem mean average compared to the control mean average. This contrasts with findings in Stevia rebaudiana ‘Bertoni’, where flurprimidol increased xylem thickness by 27.1% [30]. Suppressive effects have also been reported; uniconazole reduced xylem cell bands by 65% in treated plants [31], likely due to the limited xylem mobility of triazole compounds [32,33]. Similarly, Syzygium campanulatum showed a 48.6% reduction in xylem thickness following triazole application [34].

In passion fruit, paclobutrazol increased vessel density by 38% but reduced overall xylem diameter by 44.4%, likely through enhanced cambial differentiation coupled with inhibited cell elongation [26,35]. The treatment of IBA has also demonstrated positive effects on xylem development, as seen in thicker xylem bands of Corymbia torelliana × C. citriodora seedlings [36]. The general trend of reduced xylem width observed here may reflect GA inhibition via triazoles, but a lack of consistent response across treatments suggests possible sweetpotato-specific hormone sensitivity or interaction effects. Although minor differences were noted in the present study, future work should focus on optimal rates and chemical combinations that elicit meaningful changes in xylem structure. Increased xylem thickness can improve water and nutrient transport, drought resilience, and disease resistance, all of which are relevant to enhancing sweetpotato productivity [9,37].

5. Conclusions

This study provides novel insights into how foliar-applied plant growth regulators (PGRs) influence the internal anatomical structure of sweetpotato slips, with a focus on four tissue types—epidermis, collenchyma, parenchyma, and xylem. Among the treatments evaluated, flurprimidol at 120 mg·L⁻¹ significantly increased epidermal thickness by 31.8%, indicating its potential to enhance structural resilience in the outermost stem tissue. Conversely, paclobutrazol at 30 and 60 mg·L⁻¹ significantly decreased collenchyma thickness, suggesting a possible inhibitory effect on support tissue development at those concentrations. Other treatments showed minor, non-significant trends, highlighting the complexity of hormonal interactions in sweetpotato stem development.

The anatomical changes observed—particularly in the epidermis—could have practical implications for improving slip transplant success under field conditions. A thicker epidermis may confer enhanced resistance to desiccation, mechanical injury, and pathogen invasion during the critical establishment phase. This trait is especially valuable for greenhouse-produced slips, which often lack structural robustness due to protected growth environments.

Despite the limited number of statistically significant results, the findings emphasize the tissue-specific and concentration-dependent nature of PGR effects in sweetpotato. The variation in anatomical responses across treatments underscores the need for further investigation into optimal PGR formulations, application rates, and timing. Additionally, the lack of consistent trends in parenchyma and xylem tissues suggests that certain stem structures may be less responsive to exogenous hormone treatments, and different approaches may be required.

Ultimately, this research contributes to a growing body of evidence supporting the strategic use of PGRs in vegetative propagation systems. Enhancing internal stem structure represents a promising avenue for improving sweetpotato transplant quality and field performance, with broader implications for other vegetatively propagated crops.