Sequential Application of 1-naphthalenacetic Acid and Putrescine Promotes the Developmental Growth of Adventitious Roots in Chrysanthemum Cuttings

Abstract

Chrysanthemums are among the most popular and economically valuable ornamental plants in the world. Herein, we investigated the effects of diamine putrescine (Put), a polyamine involved in plant cell differentiation and stress resistance, and 1-naphthaleneacetic acid (NAA), an auxin widely considered essential for root initiation, on the development of adventitious roots (ARs) and root systems in chrysanthemum cuttings. We also determined the optimal combination of concentrations of these treatments for AR developmental growth. When the cuttings were cultured with different concentrations of Put solutions, clear increases in AR length were recorded, although the number of ARs was unaffected. In addition, the number of lateral roots arising from ARs was increased by Put application. Treatment with 5-mg·L⁻¹ NAA in the first week followed by 100-mg·L⁻¹ Put in the next three weeks considerably increased the AR length compared with the use of each reagent alone. With the sequential application of 5-mg·L⁻¹ NAA and 200-mg·L⁻¹ Put, the fresh weight of the total roots that developed from the cuttings was increased by 6.05-fold compared with that without treatment. The superoxide dismutase activity and superoxide anion production rate were reduced in roots developed from Put-treated chrysanthemum cuttings. These results may indicate that Put application affected AR elongation by altering the quantitative balance among reactive oxygen species.

Introduction

Chrysanthemum (Chrysanthemum morifolium) is a widely cultivated and economically important ornamental plant globally. When chrysanthemums are agriculturally produced, they are usually propagated by taking stem cuttings from stock plants. An adventitious root (AR) first emerges from the base of the cuttings, then grows along with lateral root formation, and is finally distributed in the soil. During the developmental process, various environmental factors, such as light, temperature, and mineral nutrition, affect AR growth (Druege et al., 2016; Mhimdi and Perez-Perez, 2020; Steffens and Rasmussen, 2016). In chrysanthemum, it is well known that the rooting rate is relatively high when cuttings are planted in cell trays for seedlings. On the other hand, the method of planting the cuttings directly in the field may result in a lower rooting rate or require treatment to prevent desiccation, depending on the cultivar and cropping type. Similarly, in vegetatively propagated plants other than chrysanthemum, low temperature delays rooting and prolongs the cultivation period before rooting, which limits the season for taking cuttings. Therefore, there is a need for novel reagents and application methods that are broadly effective for horticultural plants and that better promote AR formation and elongation.

AR development is promoted by auxins, such as indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) (Gonin et al., 2019; Lakehal and Bellini, 2019; Pacurar et al., 2014). In petunia cuttings, transient increases in IAA accumulation and the induction of auxin-responsive GH3 expression occur at the base of the stem after excision (Ahkami et al., 2013). Similarly, in tomato cuttings, IAA accumulates at the bottom of excised stems before AR formation (Guan et al., 2019). Consistent with these findings, numerous studies have revealed that exogenous auxin treatment increases the number of ARs that develop from cuttings in different species (Druege et al., 2016; Wei et al., 2019). In fact, IBA solution and 1-naphthylacetamide, a precursor of 1-naphthaleneacetic acid (NAA), are widely used by farmers as reagents to treat cuttings to promote rooting. It has also been shown that the establishment of an auxin gradient dependent on polar transport is essential for AR formation (Ahkami et al., 2013; Garrido et al., 2002; Lin and Sauter, 2019). Thus, intracellular quantitative changes and the gradient of auxin accumulation in the rooting zone are crucial for AR formation in cuttings, whereas several studies have reported that long duration exogenous auxin treatment inhibits root elongation (De Klerk et al., 1999; Eliasson et al., 1989; Naija et al., 2008). These findings indicate that the optimal concentration and timing of auxin application for promoting AR initiation and elongation are species-dependent.

Polyamines are low-molecular-weight basic compounds present in almost all cells of living organisms (Tabor and Tabor, 1984). The major cellular polyamines are diamine putrescine (Put), triamine spermidine, and tetraamine spermine. Within cells, polyamines bind to and stabilize various organic macromolecules, including DNA, RNA, phospholipids, and proteins (Igarashi and Kashiwagi, 2010; Schuster and Bernhardt, 2011). Polyamines play multiple roles in cell proliferation, growth, differentiation, senescence, and apoptosis and confer abiotic stress tolerance in plants (Chen et al., 2019; Igarashi and Kashiwagi, 2010). Exogenous auxin application to the rooting of walnut and poplar cuttings increased endogenous Put levels (Hausman et al., 1995; Heloir et al., 1996). In addition, the exogenous application of an inhibitor of polyamine biosynthesis and some analogs of polyamine precursors inhibited root formation in mung bean hypocotyls, suggesting that Put accumulation or polyamine biosynthesis is required for AR formation (Friedman et al., 1982). Furthermore, polyamines promoted root elongation by increasing root cell division in regenerated Virginia pine plantlets (Tang and Newton, 2005). As polyamines are closely involved in AR development together with auxin, we hypothesized that polyamines could promote AR development from chrysanthemum cuttings and facilitate their distribution in the soil.

In this study, using chrysanthemum, which has a synchronous rooting period and for which the effects of exogenously applied reagents can be studied in a relatively short time, we tried to identify novel reagents that could promote rooting and AR elongation using the number and the growth of ARs as indicators. Likewise, we analyzed the effects of NAA and Put application on superoxide dismutase (SOD) activity and the superoxide anion (O₂^•−) production rate and their relationship with rooting capacity.

Materials and Methods

Plant materials and growth conditions

The parent stocks of C. morifolium ‘Kaori’ used herein were provided by Yamamoto Farm Co., Ltd., Hiroshima, Japan. They were cultivated in an experimental field (34°N and 132°E) of the Hiroshima Institute of Technology (Hiroshima, Japan). Excised stem cuttings harboring 8–10 leaves of similar size were rooted in seedling trays (5 × 5 × 5 cm³) with vermiculite for rooting experiments. These cuttings were grown for up to four weeks under a 12-h/12-h photoperiod (50–60 μmol·m⁻²·s⁻¹ illumination) at 25°C ± 1°C.

Chemical applications in rooting experiments

The NAA and Put stock solutions (1,000×) were prepared by dissolving NAA (Fujifilm Wako Pure Chemical, Osaka, Japan) in ethanol and putrescine dihydrochloride (Fujifilm Wako Pure Chemical) in ultrapure water. The NAA stock solution was diluted in water to 5 or 10 mg·L⁻¹ with 0.1% (w/v) ethanol and irrigated onto vermiculite for the first week of cultivation after preparing leaf cuttings. Similarly, the Put stock solution was diluted in water to a concentration of 100–400 mg·L⁻¹ and irrigated onto vermiculite at weeks 2–4 of cultivation. These diluted solutions were applied every three days at approximately 30 mL per cell. The method, amount, and frequency of application of the NAA and Put solutions were determined based on preliminary experiments, which confirmed that the result was reproducible for rooting and AR growth without inhibiting plant growth. The NAA solution was applied to the soil of the cells every three days in the same manner as the application of Put solution.

Root growth measurements

Rooted cuttings after four weeks of cultivation with chemical application were used for root growth measurements. To measure their number and length, ARs (>1 cm) that emerged from the basal part of stems were counted and measured. To measure root biomass, entire fresh roots were individually weighed. Lateral root densities were measured using a previously published method (Gonzalez-Hernandez et al., 2020).

Measurements of superoxide dismutase activity and the superoxide anion production rate

One hundred milligrams fresh weight of roots that developed from the cuttings were homogenized in 1 mL of 50 mM phosphate buffer saline (PBS) (pH 7.8), and the homogenates were centrifuged at 12,000 rpm at 4°C for 20 min. The supernatants were transferred to new tubes as crude extracts for further use. The SOD activity and the O₂^•− production rate were determined using the crude extract according to a previous study (Qi et al., 2019) with some modifications.

Briefly, to measure the SOD activity, 50 μL of the crude extract was mixed with 1.5 mL of PBS (50 mM, pH 7.8), 0.3 mL of methionine (130 mM), 0.3 mL of nitrotetrazolium blue chloride (750 μM), 0.3 mL of ethylene diamine tetraacetic acid (100 μM), and 0.25 mL of sterile water. Finally, 0.3 mL of vitamin B2 (20 μM) were added to the reaction mixture. After 45 min of incubation at 25°C under a light intensity of 50–60 μmol·m⁻²·s⁻¹, the reaction was stopped by switching off the light and the absorbance of the reaction at 560 nm was measured.

To measure the O₂^•− production rate, 0.3 mL of the crude extract were mixed with 0.7 mL of PBS (50 mM, pH 7.8) and 1 mL of hydroxylamine hydrochloride (10 mM). Then, the reaction solution was incubated at 25°C for 1 h. The solution was further mixed with 1 mL of 4-aminobenzenesulfonic acid (17 mM) and 1 mL of alpha-naphthylamine (7 mM). After incubation for 20 min at 25°C, the absorbance of the reaction at 530 nm was measured.

The absorbance at 530 and 560 nm was measured using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). All chemicals used to measure the SOD activity and the O₂^•− production rate were purchased from Fujifilm Wako Pure Chemical.

Statistical analyses

The data are expressed as the mean ± standard error of at least five independent samples. Statistical significance was calculated using the Student’s t-test with Bonferroni’s correction performed only after ANOVA indicated that not all means were equal (P < 0.05). All data were analyzed by a two-way ANOVA with the NAA and Put application as main effects and their interaction effect (P < 0.05). All statistical analyses were performed using the free software environment R version 4.0.2.

Results

Effect of NAA and Put applications on the number and lengths of adventitious roots of chrysanthemum cuttings

Under our culture conditions, the emergence of ARs in chrysanthemums was clearly observed at two weeks after cutting (Fig. 1A, B). Numerous lateral roots developed from ARs at four weeks after cutting (Fig. 1C). At the base of the cuttings, callus formation was observed approximately three days after cutting and bulges corresponding to AR primordia were observed at seven days after cutting (Fig. 1D–F). At approximately 10 days after cutting, AR emergence was observed at the basal part of the stems. To investigate the effect of Put and the sequential application of auxin and Put on AR development in chrysanthemum cuttings, different concentrations of auxin and Put solutions were applied to the cuttings and any morphological changes were observed. Although the magnitude of the effect of exogenous auxin application is likely to vary between species, in the case of C. indicum, NAA has been shown to contribute to an increase in shoot fresh weight compared to IAA and IBA at low treatment concentrations (Ghimire et al., 2022). In this experiment, therefore, NAA solution was applied to the culture soil through the first week after pinching a leafy shoot to synchronously induce the formation of AR primordia, with Put applied for the following three weeks. Furthermore, we measured the number and length of roots after sequential application of different concentrations of NAA and Put to investigate their effects.

Fig. 1

Morphological changes in chrysanthemum stem cuttings. (A–C) Gross morphology of the cuttings immediately after excision from the parent stock (A), after two weeks of cultivation (B), and after four weeks of cultivation (C). (D–F) Base of stem cuttings cultured for three (D), seven (E), or 10 days (F) after excision from the parent stock. Callus formation was observed three days after excision (D), and AR primordia were observed seven days after excision (E). ARs developed at the basal part of the stems 10 days after excision (F). Arrows indicate the calluses, AR primordium, and AR in (D)–(F), respectively. Scale bars, 1.5 mm.

First, we examined the morphological characteristics of ARs in the absence of Put application and observed that the number of ARs was considerably increased by 5- and 10-mg·L⁻¹ NAA application (Fig. 2A). Conversely, in the absence of NAA, Put application did not significantly alter the number of ARs. Next, we focused on the length of ARs, which was not affected by NAA in the absence of Put (Fig. 2B). However, in cuttings treated with more than 100-mg·L⁻¹ Put, AR length was significantly increased in the absence of NAA or 5-mg·L⁻¹ NAA. There was no further increase in AR length in cuttings treated with 200-mg·L⁻¹ or 400-mg·L⁻¹ Put compared to the 100-mg·L⁻¹ Put application. Notably, the sequential application with 5-mg·L⁻¹ NAA and 100-mg·L⁻¹ Put significantly increased the AR length compared to results obtained from cuttings treated with 100-mg·L⁻¹ Put alone.

Fig. 2

Assessment of AR development in stem cuttings cultured with different concentrations of NAA and Put. (A) Number and (B) length of ARs developed from stem cuttings. Bars represent mean ± SE (n = 5). Significance determined by Bonferroni’s test (P < 0.05) is indicated by different letters.

Effect of sequential applications of NAA and Put on the fresh weight of roots and lateral-root development in chrysanthemum cuttings

To compare the root biomass developed from stem cuttings treated with Put and/or NAA, the fresh weights of the total roots were measured. In the absence of NAA, a significant difference was observed between 400-mg·L⁻¹ Put and mock application cuttings (Fig. 3A). In cuttings treated with 5-mg·L⁻¹ NAA, the effects of more than 100-mg·L⁻¹ Put application on fresh weight were clearly evident. Cuttings sequentially treated with 5-mg·L⁻¹ NAA and 200-mg·L⁻¹ Put exhibited a 6.05-fold increase in the fresh weight compared with untreated cuttings. Compared to the 5-mg·L⁻¹ NAA application, the effect of Put on increase in the fresh weight of roots was smaller in cuttings treated with 10-mg·L⁻¹ NAA. The effect of 5-mg·L⁻¹ NAA on the increase in fresh weight of total roots at 400-mg·L⁻¹ Put was not significant, in contrast to that of 100-mg·L⁻¹ and 200-mg·L⁻¹ Put. Two-way ANOVA verified that 5-mg·L⁻¹ NAA application significantly increased the fresh weight with the synergistic interaction of application of 100-mg·L⁻¹ and 200-mg·L⁻¹ Put (P < 0.05). In addition, we focused on the lateral roots that secondarily developed from ARs. Figure 3B shows the gross morphology of cuttings cultured under various combinations of NAA and Put solutions at different concentrations. Numerous short lateral roots appeared to be densely packed between ARs. Therefore, we compared the lateral root densities between cuttings treated with 100-mg·L⁻¹ Put and mock-treated cuttings. The lateral root density was calculated by dividing the number of lateral roots generated from an AR by the AR length. The results demonstrated that the lateral root density was increased by approximately 1.51-fold in the 100-mg·L⁻¹ Put-treated cuttings compared with that in the mock-treated cuttings (Fig. 3C, D).

Fig. 3

Changes in the biomass of ground tissue and growth of lateral roots developed from ARs in stem cuttings following application of different concentrations of NAA and Put. (A) Fresh weight of total roots developed from stem cuttings. Bars represent mean ± SE (n = 5). Significance determined by Bonferroni’s test (P < 0.05) is indicated by different letters. (B) Gross morphology of stem cuttings cultured with different concentrations of NAA and Put. (C) Lateral root density of cuttings cultured with the mock control and Put (100 mg·L⁻¹). Bars represent mean ± SE (n = 5). An asterisk indicates a significant difference (Student’s t-test, *P < 0.05). (D) Lateral roots developed from ARs. The left panel presents ARs cultured with a mock control, and the right panel presents ARs cultured with Put (100 mg·L⁻¹). Scale bars, 2 cm.

Effect of Put application on SOD activity and the superoxide anion production rate in chrysanthemum cuttings

Several studies have illustrated that root growth is regulated by reactive oxygen species (ROS) such as O₂^•− and hydrogen peroxide (Mase and Tsukagoshi, 2021; Singh et al., 2016). To determine whether the Put treatment altered the state of oxidative stress in AR cells generated from chrysanthemum cuttings, we examined the activity of SOD, which catalyzes the removal of O₂^•−, a type of ROS, to produce hydrogen peroxide. No substantial change in the SOD activity was observed in the presence of NAA (Fig. 4A). In contrast, mean SOD activity was significantly reduced by Put application in the absence of NAA, with the 400-mg·L⁻¹ Put application reducing the SOD activity by an average of 22.4% compared to the control value. The production rate of O₂^•−, a substrate for the reaction catalyzed by SOD, was measured and compared among the roots generated from each cutting. Similar to the results for the SOD activity, no significant change in the O₂^•− production rate was detected by NAA application, whereas the production rate was significantly reduced by applications at Put concentrations above 100 mg·L⁻¹ (Fig. 4B). The lowest O₂^•− production rate was detected in cuttings sequentially treated with 5-mg·L⁻¹ NAA and 200-mg·L⁻¹ Put.

Fig. 4

Changes in SOD activity (A) and O₂^•− production rates (B) in the ground tissues of rooted cuttings treated with different concentrations of NAA and Put. Bars represent mean ± SE (n = 5). Significance determined by Bonferroni’s test (P < 0.05) is indicated by different letters.

Discussion

In this study, Put application to chrysanthemum cuttings increased the length of ARs emerging from the base of the cuttings, although it did not increase the number of ARs as induced by NAA application (Fig. 2A, B). The stimulatory effect of Put on root elongation shown in these results is consistent with the findings that exogenous application of polyamines promoted root elongation in regenerated Virginia pine plantlets and alleviated root growth inhibited by a DNA-damaging agent that induced interstrand cross-linking (Tanaka et al., 2019; Tang and Newton, 2005). These results indicate that polyamine application can promote root elongation under certain conditions, depending on the plant species and tissue. Although only the diamine Put was used in this study, it is expected that triamine and tetraamine (spermidine and spermine, respectively) could also be applied to cuttings to more strongly promote the growth of the ARs. This study is the first to show that polyamines have the potential to be agents that promote the establishment of roots in horticultural plant cuttings.

Our results showed that one week of treatment with 5-mg·L⁻¹ NAA followed by 200-mg·L⁻¹ Put treatment for three weeks resulted in a 6.05-fold increase in the fresh weight of the total roots (Fig. 3A). This change in fresh weight of roots is likely due to a combination of two effects: an increase in the AR number due to NAA application and an increase in AR length due to Put application. The effect of NAA application on the increase in fresh weight of roots was particularly large in cuttings treated with 5-mg·L⁻¹ NAA 100–200-mg·L⁻¹ Put, suggesting that other factors for the increase in the fresh weight of roots may include enhanced lateral root growth and increased water content in root tissue (not tested in this experiment). In contrast, the effect of NAA on the increase in the root fresh weight was reduced when Put was applied at a high concentration of 400 mg·L⁻¹ (Fig. 3A). This decrease was also observed for the number of ARs, with cuttings treated with 400-mg·L⁻¹ Put showing a weaker NAA effect on increasing the number of ARs. Since it has been reported that IAA accumulation is suppressed when high concentrations of Put are sprayed on chrysanthemums (Xu et al., 2014), it is possible that application of high concentration Put of 400 mg·L⁻¹ reduced the amount of endogenous auxins, which may have attenuated the effect of NAA application on AR number and root fresh weight. It is also well known that in several plants, excessive doses of auxin inhibit growth (De Klerk et al., 1999; Eliasson et al., 1989; Yan et al., 2014), so it is convincing that the most effective dose for fresh weight of roots in this study was 5-mg·L⁻¹ NAA rather than 10-mg·L⁻¹ NAA.

We found that Put application on chrysanthemum cuttings reduced the SOD activity in roots developed from the cuttings (Fig. 4A). In Virginia pine, exogenous application of Put has been reported to induce diamine oxidase (DAO) activity, an enzyme that catalyzes the oxidative deamination and degradation of Put and generates hydrogen peroxide, in a concentration-dependent manner (Bagni and Tassoni, 2001; Tang and Newton, 2005). It has also been shown in pea root nodules that DAO activation leading to overproduction of hydrogen peroxide promotes root cell elongation (Sujkowska-Rybkowska and Borucki, 2014). Given that hydrogen peroxide has also been shown to promote cell elongation in the elongation zone in Arabidopsis (Tsukagoshi, 2012; Tsukagoshi et al., 2010), it is possible that high concentration Put application (above 100 mg·L⁻¹) on chrysanthemum cuttings caused overaccumulation of hydrogen peroxide in the tissue, which may have promoted root cell elongation, but also led to a decrease in SOD activity to maintain cellular homeostasis. On the other hand, it is interesting to note that Put application reduced the production rate of O₂^•−, a substrate of the SOD catalytic reaction, as well as reducing SOD activity (Fig. 4B). However, it is well known that polyamines act as free radical scavengers and contribute to oxidative stress tolerance in several type of cells (Ha et al., 1998; Murray Stewart et al., 2018; Solmi et al., 2023), and in tobacco protoplasts, polyamine application has also been found to inhibit NADPH oxidase-mediated O₂^•− generation (Papadakis and Roubelakis-Angelakis, 2005). Based on these findings, we speculate that the two results, decreased SOD activity and a reduced O₂^•− production rate, may reflect independent effects of Put application, i.e., suppression of SOD activity possibly by overproduction of hydrogen peroxide and inhibition of free radical production, respectively. Future detailed measurements of hydrogen peroxide accumulation in ARs developed from Put-treated chrysanthemum cuttings are expected to provide new insights into the complicated quantitative changes in ROS that occur in Put-treated plant tissues.

In this study, our experiments were conducted by watering cuttings with NAA solution every days. In agricultural production, cuttings are often soaked in auxin solution once before planting, so fine-tuning the appropriate concentrations and application methods for auxin and Put should be considered in the future for practical application of our results.

Acknowledgements

The authors would like to thank Enago (www.enago.jp) for the English language review.

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