Seaweed Extracts as Substitutes of Synthetic Hormones for Rooting Promotion in Rose Cuttings

Abstract

In the horticultural sector, the achievement of an efficient and eco-friendly sustainable production of plants is nowadays challenging. Indeed, in plant vegetative propagation of woody ornamentals, the substitution of chemical products used to promote rooting of cuttings with natural extracts would be a desirable goal. Thus, the aim of this work was to test the replacement of synthetic phytoregulators, such as auxins and brassinosteroids, with biostimulants, such as seaweed extracts, for the rooting promotion of rose cuttings. The rooting rate and biometric parameters of control cuttings treated with distilled water were compared with those of cuttings treated with synthetic hormones, i.e., auxins or 22(S),23(S)-homobrassinolide, or two commercial products based on low temperature seaweed extracts, i.e., Kelpak^® and Phylgreen. Two scented hybrid tea rose cultivars were used to assess possible genotype-dependent effects, i.e., ‘Michelangelo^®’ and ‘Cosmos^®’. Auxins confirmed their role in root growth enhancement in ornamental plant cuttings. Like these phytoregulators, Kelpak^® improved the survival rate and root biometric parameters of both rose cuttings, highlighting its suitability for the replacement of synthetic products used for rooting promotion in rose propagation. Brassinosteroids showed a species-dependent effect, increasing the root biomass in ‘Cosmos^®’ while it resulted as distilled water in ‘Michelangelo^®’. Phylgreen did not improve the rooting of both rose cuttings, highlighting the necessity of evaluating the applicability and methodology for this product before its use. In conclusions, our results highlighted the possibility to replace chemical products in rose cutting production.

1. Introduction

A key challenge in the production of ornamental plants is the combination of sustainable practices and cultivation and propagation efficiency [1]. Plant propagation in the floricultural sector relies on different techniques, among which the use of stem cuttings is one of the most used for woody plant species, such as roses, also for its timesaving and cost-efficiency [2,3]. This method allows the maintenance of desirable characteristics in superior rose cultivars, particularly considering their high degree of heterozygosity and polyploidy, but can be affected by the deep genotypic differences in rooting ability within this genus [4]. Indeed, in stem cutting propagation, adventitious root formation is a key step to be successful and it is promoted by producers in nurseries with the application of phytoregulators [5,6]. The most widely spread commercial products consist of auxins such as indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) [7]. Other phytoregulators have been shown to have a role in rooting promotion [8], but they are poorly investigated for commercial applications. Among them, brassinosteroids (BRs) are plant-specific steroid hormones that have a role in both growth promotion, including root formation, and abiotic stress tolerance [9]. Indeed, BRs increased the degree of rooting and root length as well as the total soluble sugars in stem cuttings of two cultivars of barberry ornamental plants [10]. However, the small amount and fast metabolization of BRs in plant tissues make the production costs of natural compounds too high, and therefore structural and functional analogues are commonly used, such as the 24-epibrassinolide [11].

In modern agriculture, the substitution of chemical inputs, such as phytoregulators, with alternative natural eco-friendly products presents a key challenge [12] also to meet the transition towards agroecological production systems and organic farming principles [13]. In this context, seaweed extracts constitute a promising alternative solution to the use of synthetic products in the promotion of plant yield and health [14]. Different seaweed species have shown their activity as biostimulants, probably related to the presence of bioactive ingredients such as phytohormones as well as carbohydrates, proteins, and mineral elements [15,16]. Moreover, seaweed extracts have been shown to be not only naturally enriched in phytohormones but also able to promote the endogenous biosynthesis of auxins, cytokinin, and gibberellins [17]. Beyond their effect as growth promoters, seaweed extracts have also disease suppressing effects [18], potentially reducing the application of phytochemicals as well. However, the molecular mechanisms behind their function are still little known. Moreover, new sustainable extraction procedures have been developed to avoid the use of chemicals and preserve the seaweed’s original quality, increasing their environmentally friendly aspects [18]. Despite these promising features, to our knowledge, only a few seaweed extracts have been tested for rooting promotion in rose cutting propagation, prevalently containing the Ascophyllum nodosum extract [6].

Thus, the aim of this work was to test the replacement of synthetic phytoregulators with two seaweed extracts, i.e., Kelpak^® and Phylgreen, for promoting the rooting of rose cuttings. The two seaweed products were evaluated in comparison with the absence of treatment (only distilled water), or two different synthetic phytoregulators, i.e., auxins or BRs. The extracts were tested on two rose cultivars to assess possible differences related to genotype-dependent responses.

2. Materials and Methods

2.1. Plant Material and Treatments

The newly produced young herbaceous stems were sampled at the end of March 2021, at the beginning of the growing season, from one-year old plants of two Rosa spp. cultivars, ‘Michelangelo^®’ and ‘Cosmos^®’, both scented hybrid tea Meilland (Meilland International, Le Luc-en-Provence, France), maintained within a greenhouse and pruned on 11 February 2021. Rose plants were fertigated with a nutrient solution containing 1.5 g L⁻¹ of Peters^® Excel CalMag Finisher (13-5-20 + 7CaO + MgO + TE, ICL Specialty Fertilizers, Tel Aviv, Israel), maintaining the pH at 5.5–6.0. Single node stem cuttings (3.5–4.5 cm) two weeks old were collected, leaving a half leaf portion to assure transpiration on 29 March. Five different treatments were applied to both cultivar cuttings (90 cuttings for each treatment and each cultivar for a total of 900 cuttings): (1) distilled water (H₂O); (2) 4000 ppm of indole-3-butyric acid and 1-naphthaleneacetic acid (Merck KGaA, Darmstadt, German) in distilled water (AUX); (3) 5 ppm of 22(S),23(S)-homobrassinolide (Merck KGaA, Darmstadt, German) in distilled water (BRA); (4) 10% Kelpak^® (Agricola Internazionale S.R.L., Pisa, Italy) in distilled water (KEL); (5) 10% Phylgreen (Tradecorp Italia & South East Europe, Bologna, Italy) in distilled water (PHY). Kelpak^® contains an Ecklonia maxima extract obtained through a low temperature mechanical extraction avoiding the use of chemicals and without altering the original product. It has auxins and cytokinins in an optimal balance for rooting promotion. Phylgreen is made from A. nodosum and, as the Kelpak^®, it is also obtained through a low temperature mechanical extraction without chemicals, preserving the product features.

Treatments were applied through the immersion of basal cutting portions (1 cm) for 5 s (H₂O, AUX, BRA) or 1 min (KEL and PHY) within each specific solution. After the treatments, cuttings were immediately placed in holes filled with the substrate (1:1 v/v of peat and perlite) and irrigated. Cuttings were sprayed with water 5 times per day for 3 min for 3 weeks, while after this period they were sprayed 3 times per day for 3 min. Average temperature, humidity, and lighting during the experimental period were 21 °C, 70%, and 322 Watt m⁻². Data were acquired by an EnviroMonitor weather station (Davis Instruments, Hayward, CA, USA).

2.2. Biometric Measures

After 14 weeks from the transplant (30 June), the number of cuttings was evaluated to establish the rooting rate percentage for each treatment. Nine cuttings were sampled and used as biological replicates for the biometric measures. In particular, the shoot number, shoot length, and root fresh weight (FW) were evaluated. Root images were acquired, and root length and area were measured through Fiji software. Roots were then dried at 75 °C for 2 days and the dry weight was measured (DW).

2.3. Carbohydrates Analysis

Since a higher soluble sugar concentration has been reported to match with better rooting performance in rose cuttings [19], carbohydrates were also quantified in stems during the rooting period.

For each treatment, 12 cuttings of each cultivar were sampled at the beginning of the trial and 4 and 16 days after the treatments. The bark with the emerging callus was collected, frozen in liquid nitrogen, and stored at −80 °C for carbohydrate analysis. The bark was reduced in powder with liquid nitrogen and used for the determination of soluble sugars through the Anthrone colorimetric method (Merck KGaA, Darmstadt, Germany), reading the absorbance at 620 nm against a standard curve of glucose.

2.4. Statistics

Data were tested for normal distribution using the Shapiro–Wilk normality test and then analyzed by a two-way ANOVA (treatment and cultivar as variables) to assess differences in treatment between the cultivars and then with a one-way ANOVA, followed by a Tukey’s post-hoc test, to highlight significant differences between treatments (p ≤ 0.05, 0.01, and 0.001). The statistical analyses and graphs were performed with Prism 9 (GraphPad Software, Inc., La Jolla, CA, USA).

3. Results

3.1. Survival Percentage and Biometric Measures

Control cuttings of both cultivars treated with distilled H₂O showed a rooting percentage on average of 65% (Figure 1). Cuttings treated with BRA and PHY had a slightly lower rooting rate than control cuttings in cv. ‘Michelangelo^®’. The highest rooting percentage in this cultivar was measured under KEL treatment (77%, + 19% in comparison to control cuttings). On the contrary, all treatments increased the rooting percentage in cv. ‘Cosmos^®’, particularly the cuttings treated with AUX, which had a survival rate 22% higher than the control ones.

After 16 days from the treatments, callus formation was mostly present in the lowest part of cuttings treated with AUX, BRA, and KEL (Figure 2). Callus was absent from cuttings treated with PHY, while it was a small portion in those treated with H₂O.

Every cutting produced one single sprout and sprout length was not significantly different between treatments in both cultivars (Figure 3).

Treatments had a different effect on the root biometric parameters of the two rose cultivars (Figure 4). The root FW (Figure 4A) was higher under AUX and KEL treatments in cv. ‘Michelangelo^®’ (+99 and 62%, respectively) and under BRA treatment in cv. ‘Cosmos^®’ (+82%). The same trend was shown in root DW (Figure 4B) by cv. ‘Cosmos^®’. Root length (Figure 4C) was higher under AUX and KEL treatments in cv. ‘Michelangelo^®’ (+103 and 75%, respectively) and under BRA and PHY treatments in cv. ‘Cosmos^®’ (+76 and 71%, respectively). Root area (Figure 4D) was higher under AUX, KEL, and PHY treatments in cv. ‘Michelangelo^®’ and under AUX, BRA, and PHY treatments in cv. ‘Cosmos^®’.

3.2. Soluble Sugars

Total soluble sugars at the beginning of the trial and after 4 and 16 days from the treatments are reported in Table 1. At the beginning of the trial, the amount of soluble sugar was the highest measured, and it was similar between the two cultivars. Four days after the treatments, the soluble sugar content was lower in both cultivars, and it was not significantly different between the treatments. After 16 days from the treatments, the soluble sugar content was lower than the previous days in all treatments and, moreover, it was lower under PHY treatment than in control cuttings in cv. ‘Michelangelo^®’.

4. Discussion

The rose is one of the most important and valuable ornamental shrubs worldwide [20]. Its propagation is usually achieved by cuttings with a rooting efficiency varying from 0 to 100% [4]. The cvs. ‘Cosmos^®’ and ‘Michelangelo^®’ showed a rooting percentage of about 65%, highlighting a medium rooting ability. The possible failures in root formation are usually overcome by producers with the application of plant growth regulators [3]. In particular, the rooting promotion of cuttings using auxins is widely used in commercial plant propagation [7]. Indeed, AUX treatment showed an increase in survival percentage, callus formation, and root biometric parameters in both cultivar cuttings in comparison with the control conditions, i.e., only distilled water. Other authors reported an increase from 33 to 65% in rooting of R. centifolia medial cuttings in the same substrate using 3500 ppm of auxins [21] or from 67 to 75% or 81% in rooting of R. damascena cuttings in sand using 200 mg dm⁻³ IAA or 25 mg dm⁻³ of NAA, respectively [22]. Thus, the aim of this work was to find alternative substances to auxins for the promotion of cutting rooting suitable for rose propagation.

BRs have already shown their role in rooting promotion of ornamental plant cuttings [10], but commercial synthetic products are still based principally on auxins and, to our knowledge, they have never been tested on rose cuttings. BRs have shown contrasting effects on rooting in woody cuttings of different plant genotypes [23,24]. In addition, in our experimental conditions, 22(S),23(S)-homobrassinolide showed opposite effects on the two rose cultivars. The cuttings of cv. ‘Michelangelo^®’ treated with BRA had a similar behavior to those treated with only distilled water, while in cv. ‘Cosmos^®’ this treatment showed the best results in terms of root biometric parameters. Thus, BRs were found to be unsuitable for propagation in operational conditions since testing on every specific rose genotype is required. The worst performance in rooting under BRA treatment retrieved in cv. ‘Michelangelo^®’ matched with a low amount in soluble sugars within the emerging callus 16 days after the treatment. Since a positive correlation between root number and soluble sugar concentration was reported in other rose cultivar cuttings [19], this result might support a less susceptibility of cv. ‘Michelangelo^®’ to rooting under BRA treatment. Indeed, other authors also highlighted that a depletion of carbohydrates reduced callus and root formation in single-node leafy stem cuttings [25]. However, a constitutional decrease in soluble sugars during the first 15 days has already been reported within the bark of cuttings of Zizyphus jujuba Mill. [26] and could explain the lower level of soluble sugars found in all rose cuttings in comparison with the beginning of the trial.

Biostimulants like seaweed extracts seem to be a promising alternative to synthetic products in horticulture [14]. Seaweed extracts have been successfully used for the promotion of rooting in several ornamental species. Specifically, Kelpak^® has been demonstrated to promote the growth of leafy pelargonium cuttings [27]. In our experimental conditions, this natural product increased the rooting percentage and the root biometric parameters of both rose cultivar cuttings. In particular, under this treatment, the cuttings had the highest rooting percentage in cv. ‘Michelangelo^®’. Kelpak^® has been shown to contain abscisic acid, gibberellins, and BRs in higher concentrations than in kelp E. maxima tissues, and these hormones can directly contribute to the promotion of plant growth [28]. Indeed, Kelpak^® treatment increased the concentration of cytokinin and bioactive compounds in aerial parts of Eucomis autumnalis [29], as well as increased rooting rate of cuttings of Prunus marianna stockplants [30]. On the contrary, Phylgreen did not significantly improve the rooting performance of both cultivar cuttings, while in previous studies, the use of a 40% concentrated A. nodosum extract increased the rooting of Passiflora actinia by about 10% [31] and the rhizogenesis in hypocotyl slice of Prunus domestica under in vitro propagation experiments by about 30% [32]. The specific application of Phylgreen on Arabidopsis thaliana plants promoted abiotic stress resistance and growth if applied repeatedly throughout the cropping cycle [33], therefore highlighting the need to test the time and method of application. Indeed, a lower concentration of soluble sugars was measured under this treatment in cv. ‘Cosmos^®’, probably highlighting a low metabolic activity. The limited information available in the literature does not allow further argumentation on this biostimulant since contrasting results were observed on rose cuttings with respect to other investigated species. However, the results highlighted the importance of evaluating the type of seaweed to treat each specific ornamental species on the basis of the application method.

Interestingly, root DW was not particularly altered by any treatment, highlighting that the root biometric variations were principally related to increased water content and thus cell elongation. Indeed, the role of auxins in promoting cell elongation through turgor-driven processes is already known [34].

5. Conclusions

In conclusion, Kelpak^® was shown to improve both rooting percentage and root biometric parameters of both rose cultivar cuttings, highlighting its suitability for the replacement of synthetic products used for rooting promotion. BRs showed a species-dependent effect, highlighting the necessity of evaluating their applicability before their use as alternative synthetic phytoregulators to auxins. Phylgreen did not improve the rooting performance of rose cuttings, indicating that this natural product cannot be suitable for rose propagation using this methodology. Since the use of authorized hormonal agrochemicals is very limited in some countries and is getting even lower every year, this study offers a new possible product also available for organic systems. Further efforts must be conducted to clarify the biochemical and molecular mechanisms of action of this seaweed extract.

Author Contributions

Conceptualization and investigation, S.T., S.C. and B.N.; trial set up and sampling, S.T., S.C. and B.N.; formal analysis and data curation, S.T.; funding acquisition, S.C. and B.N.; Writing—original draft, S.T.; Writing—review & editing, S.T., S.C. and B.N. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

The authors would like to thank Monica Michelotti and Paolo Bini for their technical support during the trial.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Table

Figure 1. Rooting rate (%) of cv. ‘Michelangelo®’ and ‘Cosmos®’ cuttings. Values were calculated on the 900 total cuttings. Cuttings were treated with: H2O = distilled water; AUX = auxins; BRA = homobrassinolide; KEL = Kelpak®; PHY = Phylgreen.

Figure 2. Images of cuttings 16 days after the start of treatments. Orange arrows indicate the callus formation.

Figure 3. Sprout length of cv. ‘Michelangelo®’ and ‘Cosmos®’ cuttings, 14 weeks after the treatments. Cuttings were treated with: H2O = distilled water; AUX = auxins; BRA = homobrassinolide; KEL = Kelpak®; PHY = Phylgreen. Two-way and one-way ANOVA p-values are reported in the figure (ns, not significant).

Figure 4. Root fresh weight (A), dry weight (B), length (C), and area (D) of cv. ‘Michelangelo®’ and ‘Cosmos®’ cuttings, after 14 weeks from the treatments. Cuttings were treated with: H2O = distilled water; AUX = auxins; BRA = homobrassinolide; KEL = Kelpak®; PHY = Phylgreen. Two-way and one-way ANOVA p-values and Tukey’s post hoc comparisons are reported in the figure (* p < 0.05, ** p < 0.01; *** p < 0.001; ns, not significant). Letters in the figure indicate the level of difference significance.

Table 1

Soluble sugars within the bark and the emerging callus of cv. ‘Michelangelo^®’ (M) and ‘Cosmos^®’ (C) cuttings, 0, 4, and 16 days after the treatments. Cuttings were treated with: H₂O = distilled water; AUX = auxins; BRA = homobrassinolide; KEL = Kelpak^®; PHY = Phylgreen. Two-way and one-way ANOVA p-values and Tukey’s post hoc comparisons are reported in the table (* p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant).

Day	Cv.	Treatment					p
		H2O	AUX	BRA	KEL	PHY	T	C × T
0	M	18.0 ± 1.27	−	−	−	−	−	−
	C	20.8 ± 1.79	−	−	−	−	−	−
4	M	8.0 ± 0.95	7.2 ± 1.79	10.0 ± 5.60	8.8 ± 2.19	7.9 ± 3.77	ns	ns
	C	12.3 ± 2.00	14.3 ± 2.71	10.7 ± 1.23	12.6 ± 1.10	14.8 ± 4.00	ns
16	M	2.9 ± 1.16 ab	3.8 ± 1.23 a	0.3 ± 0.16 b	0.8 ± 0.82 ab	3.3 ± 1.37 ab	*	***
	C	9.7 ± 1.84 a	11.0 ± 2.19 a	8.2 ± 2.26 a	9.5 ± 2.34 a	1.6 ± 0.16 b	**

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