Foliar application of K-IBA to the Abaxial or Adaxial Leaf Surface With or Without a Surfactant Does Not Impact Root Formation in Buttonbush and Burning Bush Euonymus Cuttings¹ Open Access

Applying auxin as a foliar spray has several advantages over traditional basal application methods for rooting cuttings. These include potential worker safety and application efficiency. Foliar auxin sprays may also become important in propagation systems using automated machine-assisted sticking robotics. However, not all woody species respond to a foliar spray as well as a basal quick dip application for rooting and there are questions concerning initial auxin uptake and transport. The current study evaluated the efficacy of foliar auxin application to the adaxial (upper) or abaxial (lower) leaf surface and the impact of including a surfactant on rooting in cuttings from button bush and burning bush euonymus.

Foliar auxin application for rooting cuttings is not necessarily a new concept and was one of the auxin application methods studied as early as the 1940s (Blythe et al. 2007, Stoutemyer and O’Rourke 1945). For several reasons, the basal quick dip and talc powder application methods emerged as the preferred commercial practices for auxin application (Davies et al. 2018). There was a renewed interest in foliar auxin application in the early 2000s (Blythe et al. 2003, Blythe et al. 2004), partly due to the availability of water-soluble auxin formulations, potential for reduced worker exposure to auxin, and possible cost savings in labor efficiency (Drahn 2007, Martindell 2019).

Foliar auxin application differs fundamentally from basal auxin applications. Basal applications supply exogenous auxin directly to the site of adventitious root formation, while a foliar application relies on auxin transport from the leaf or shoot tip to the basal rooting region. Given the different modes of auxin delivery, it is understandable that selected species have responded differently to basal versus foliar applied auxin for induction of rooting in cuttings. This differential response to application method could be due to initial foliar auxin uptake or subsequent transport mechanisms. It has been assumed that the main entry of foliar applied auxin is through stomatal openings in the leaf (Koin 2009), but this has not been substantiated. Also, the epicuticle wax on the upper leaf surface could also be a barrier to auxin absorption. Therefore, the objective of the current study was to compare foliar auxin application to adaxial or abaxial leaf surfaces in comparison to basal application in two species for adventitious root formation in cuttings.

Cuttings were taken from buttonbush (Cephalanthus occidentalis) and burning bush euonymus (Euonymus alatus ‘Compactus’) plants growing on the University of Kentucky, Lexington campus.

Terminal two-node softwood cuttings of buttonbush were taken in late May. Terminal cuttings of burning bush euonymus containing an apical bud cluster and four subtending opposite leaves were taken in July or August. Cuttings were placed in plastic 606 seep standard six-pack inserts with cells 5.1 cm wide by 5.7 cm long by 8.3 cm high (2.0 in W x 2.25 in L x 3.25 in H) with a Pro-Mix BX (Premier Tech Horticulture, PA) and perlite (70:25%; v/v) substrate and rooted under intermittent mist (10 sec every 15 min) with bottom heat (25 C) (77 F) and approximate daily light integral 15 to 25 mol·m⁻²·d⁻¹. Cell packs were treated as experimental units and randomized within the mist bench for each experiment.

Untreated buttonbush or burning bush euonymus cuttings were placed directly into cell packs. One set of cuttings for both species was treated with aqueous K-IBA at 5,000 mg·L⁻¹ (210 mM) as a 3 sec basal dip. Buttonbush cuttings had K-IBA at 5,000 mg·L⁻¹ applied to each leaf on the abaxial or adaxial leaf surfaces with a small paint brush prior to sticking in cell packs. For burning bush euonymus, K-IBA at 5,000 mg·L⁻¹ with or without a surfactant at 1.0 ppm (Nu Film P, Miller Chemical and Fertilizer Corporation, Pennsylvania) was applied to the abaxial or adaxial leaf surfaces as a foliar spray application. Burning bush euonymus has a distichous leaf phyllotactic arrangement that allowed cuttings to be laid flat and 10 mL per 6 plants of K-IBA at 5,000 mg·L⁻¹ to be applied using a small hand-held atomizer to either the adaxial or abaxial leaf surface. There were 5 six-pack units per treatment (30 cuttings per treatment) and six -packs were randomly assigned within the mist bed with each six-pack considered an experimental unit. Experiments were repeated in July and August.

Leaf surfaces of burning bush euonymus were prepared from epidermal peels. A line of glue (Duro® Super Glue, Henkel Corporation, Avon, OH 44011) was spread across a glass microscope slide. A section of the abaxial or adaxial leaf surface between the midrib and margin, parallel with the midrib, was pressed against the slide and pressure was applied from the middle outward. The leaf was peeled off the slide after 60 sec leaving the epidermal peel on the slide. Peels were observed for stomatal density and photographed under a light microscope (Olympus BX40) equipped with a digital camera (Olympus DP25).

Rooting percentage and the number of roots per cutting was assessed 40 days after sticking. Mean separation was by Tukey’s test at the 5% level and percentages were transformed (square of the arcsine) where appropriate prior to statistical analysis using SigmaPlot 12.3 (Systat Software, Richmond CA).

Rooting percentages in buttonbush were between 72 and 78% for untreated cuttings and for each of the auxin application treatments (Table 1). The highest number of roots per cutting was seen in cuttings treated with a basal dip of K-IBA and the lowest rooting was in untreated cuttings. There was an approximate increase of 3.5 roots per cutting in cuttings with a foliar application of K-IBA compared to untreated cuttings and there was no significant difference between application to the adaxial or abaxial leaf surfaces.

Rooting percentages in burning bush euonymus were similar in K-IBA-treated cuttings regardless of application method or use of a surfactant (Table 2). The highest number of roots per cutting was seen in the basal dip treatment and the lowest roots per cutting in untreated cuttings. There was an approximate increase of 3 roots per cutting with a foliar spray application of K-IBA compared to untreated cuttings. There was no difference in rooting between cuttings treated on the adaxial versus the abaxial leaf surface regardless of including a surfactant.

The stomatal density was about ten times greater on the abaxial leaf surface compared to the adaxial surface (Fig. 1). The abaxial surface had approximately 165 stomata per cm⁻² compared to 15 stomata per cm⁻² on the adaxial surface.

In the species evaluated in this study, foliar-applied K-IBA appeared to enter cuttings equally well via the adaxial and abaxial leaf surfaces as evidenced by no differences in rooting percentages and roots per cutting between the two application methods (Tables 1 and 2). It has been assumed that auxin would differentially enter through the abaxial leaf surface because of reduced epicuticular wax and increased stomatal density compared to the adaxial leaf surface as observed in euonymus (Fig. 1). However, it is generally accepted that following initial severance of the cutting from the plant, stomates close and there is a concomitant reduction in transpiration and gas exchange (Loach 1988, Svenson et al. 1995, Wilkerson et al. 2005). Therefore, it might be expected that stomates would not be the major entry point for foliar-applied auxin. Additionally, most systems that deliver foliar auxin use the “spray drip down method” where the spray is applied to the adaxial leaf surface until it drips from the leaf (Kroin 2009), and this would limit auxin contact with the underside of the leaf and the majority of stomates.

If epicuticular wax or droplet adhesion to the leaf on the adaxial leaf surface presented a barrier to foliar auxin entry into the leaf, then it might be expected that inclusion of a surfactant with the auxin spray would increase uptake and subsequent rooting performance. There was no impact of a surfactant on rooting in the current study (Table 2). This is similar to foliar auxin plus surfactant studies with rose (Rosa spp.) (Blythe et al. 2004, Bowden et al. 2022), camelia (Camellia japonica L.), and magnolia (Magnolia grandiflora L.) (Bowden et al. 2022) where there was no improvement of rooting with inclusion of a surfactant. Including a surfactant is a standard practice for agronomic herbicide applications and its efficacy on leaf absorption has been shown to be influenced by environment, surfactant type and concentration, as well as plant leaf surface wax properties (Ramsey et al. 2005). It is possible that the greenhouse environment or the time between auxin plus surfactant application and the first mist events have confounded the potential positive effects of using a surfactant. Additional research may be warranted on surfactant use, especially in species that do not have a positive response to foliar auxin application.

In general, foliar auxin application has been shown to be comparable or more effective compared to basal auxin application in herbaceous and numerous woody perennial cuttings (Blythe et al. 2004, Kohler et al. 2022, Pacholczak 2015). However, in some woody perennial species basal application was shown to be superior to foliar application (Blythe et al 2004, Phillips 2017). This may be due to several factors including foliar auxin uptake, auxin transport, subsequent auxin metabolism or conjugation. Direct comparisons between foliar versus basal applied auxin on adventitious root induction can be difficult to interpret because even if the applications are made at the same auxin concentration, the amount of active auxin reaching the target cells at the cutting base may not be equivalent. In the two species used in the current study, basal application was superior to foliar-applied auxin at the same concentration, suggesting that less active auxin accumulated in the rooting region of the stem after foliar application compared to the basal dip. However, foliar application increased roots per cutting by about 20% compared to untreated cuttings for both species, demonstrating auxin uptake and transport. The concentration of 5,000 mg·L⁻¹ K-IBA used in this study was at the higher range suggested for cutting application and therefore should not have been limiting. The results suggest that differential induction of rooting between untreated, basal, and foliar auxin application may be related to foliar absorption but could also reflect the complex long-distance transport and metabolism of IBA. In apical stem cuttings, exogenous IBA, endogenous IAA, and IAA derived from ß-oxidation of IBA (Strader and Bartel 2011) could be basipetally transported following foliar IBA application. In a study using Arabidopsis thaliana L. hypocotyls, applied IBA and IAA moved basipetally at different speeds and significant amounts of IBA were converted to IAA during transport (Liu et al. 2012). It was also shown that conjugated IBA accumulated at the base preferentially to active free IBA. Future research should focus on the uptake, transport, and metabolism of IBA under cutting propagation conditions to better understand and interpret results from foliar applied K-IBA in relation to species that respond differently to application method or do not respond to foliar application.