horticulturae

Article
Seaweed Extracts as Substitutes of Synthetic Hormones for
Rooting Promotion in Rose Cuttings
Silvia Traversari 1,2 , Sonia Cacini 2, * and Beatrice Nesi 2

1 Research Institute on Terrestrial Ecosystems (IRET), National Research Council (CNR), Via Moruzzi 1,
56124 Pisa, Italy; [email protected]
2 CREA Research Centre for Vegetable and Ornamental Crops, Council for Agricultural Research and
Economics, Via dei Fiori 8, 51012 Pescia, Italy; [email protected]
* Correspondence: [email protected]

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 phytoreg-
ulators, Kelpak® improved the survival rate and root biometric parameters of both rose cuttings,
Citation: Traversari, S.; Cacini, S.; highlighting its suitability for the replacement of synthetic products used for rooting promotion in
Nesi, B. Seaweed Extracts as
rose propagation. Brassinosteroids showed a species-dependent effect, increasing the root biomass
Substitutes of Synthetic Hormones
in ‘Cosmos® ’ while it resulted as distilled water in ‘Michelangelo® ’. Phylgreen did not improve the
for Rooting Promotion in Rose
rooting of both rose cuttings, highlighting the necessity of evaluating the applicability and methodol-
Cuttings. Horticulturae 2022, 8, 561.
ogy for this product before its use. In conclusions, our results highlighted the possibility to replace
https://doi.org/10.3390/
horticulturae8070561
chemical products in rose cutting production.

Academic Editors: Jean Carlos Keywords: auxins; homobrassinolide; Kelpak® ; ornamental plant propagation; Phylgreen
Cardoso, Wagner Vendrame and
Chao Ma

Received: 9 May 2022

Accepted: 17 June 2022 1. Introduction
Published: 21 June 2022 A key challenge in the production of ornamental plants is the combination of sus-
Publisher’s Note: MDPI stays neutral tainable practices and cultivation and propagation efficiency [1]. Plant propagation in the
with regard to jurisdictional claims in floricultural sector relies on different techniques, among which the use of stem cuttings is
published maps and institutional affil- one of the most used for woody plant species, such as roses, also for its timesaving and
iations. 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
Copyright: © 2022 by the authors. step to be successful and it is promoted by producers in nurseries with the application
Licensee MDPI, Basel, Switzerland. of phytoregulators [5,6]. The most widely spread commercial products consist of aux-
This article is an open access article ins such as indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) [7]. Other
distributed under the terms and
phytoregulators have been shown to have a role in rooting promotion [8], but they are
conditions of the Creative Commons
poorly investigated for commercial applications. Among them, brassinosteroids (BRs) are
Attribution (CC BY) license (https://
plant-specific steroid hormones that have a role in both growth promotion, including root
creativecommons.org/licenses/by/
formation, and abiotic stress tolerance [9]. Indeed, BRs increased the degree of rooting and
4.0/).

Horticulturae 2022, 8, 561. https://doi.org/10.3390/horticulturae8070561 https://www.mdpi.com/journal/horticulturae

Horticulturae 2022, 8, 561 2 of 9

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 min-
eral 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 aux-
ins, 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 knowl-
edge, 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 (H2 O); (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 (H2 O, 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
Horticulturae 2022, 8, 561 3 of 9

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 differ-
ences 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 H2 O showed a rooting percent-
age 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 H2 O.
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® ’.
 ‘Cosmos
‘Cosmos
®’ (+ 82%). The same trend was shown in root DW (Figure 4B) by cv. ‘Cosmos®’.
®’ (+ 82%). The same trend was shown in root DW (Figure 4B) by cv. ‘Cosmos®’.
Root length (Figure
Root length (Figure 4C) was 4C)higher
was higher
underunder AUXKEL
AUX and andtreatments
KEL treatments
in cv. in cv. ‘Michelangelo
‘Michelangelo ®’
®’

(+ 103(+and
10375%,
and respectively)
75%, respectively) and under BRAPHY
and treatments
PHY treatments
in cv. in cv. ‘Cosmos
®’ (+ 76’ (+ 76
®
and under BRA and ‘Cosmos
and 71%, respectively). Root area (Figure 4D) was higher under AUX, KEL, and PHY PHY
and 71%, respectively). Root area (Figure 4D) was higher under AUX, KEL, and
Horticulturae 2022, 8, 561 treatments
treatments in cv. in cv. ‘Michelangelo
‘Michelangelo ®’ and ’under
® and under AUX, BRA, and PHY treatments in cv. ‘Cos-
AUX, BRA, and PHY treatments in cv. ‘Cos- 4 of 9
mos®’.mos ’.
®

H2O H2O AUX AUX BRA BRA KEL KEL PHY PHY
100 100
77 78 78
80 80 77
71
7070 7170

Rooting rate (%)

Rooting rate (%) 6966 69 70
66 63 64
63 60 6064
60 60

40 40

20 20

0 0
cv. 'Michelangelo
®' ®' cv. 'Cosmos
®' ®'
cv. 'Michelangelo cv. 'Cosmos
® ’‘Cosmos ® ’ cuttings.
FigureFigure 1.1.Rooting rate (%) of cv.
of ‘Michelangelo
Rooting ®’ and ®’ cuttings. Values were calculated on
Figure
1. Rooting rate (%) ofrate
cv. (%) cv. ‘Michelangelo
‘Michelangelo ®’ and ‘Cosmos and ‘Cosmos
®’ cuttings. Values Values
were calculatedwere
on calcu-
the 900
the 900 lated total cuttings.
on the 900
total cuttings. Cuttings
total cuttings.
Cuttings were treated
Cuttings
were treated with:
were
with: H
H2Otreated
2 O = distilled
with:water;
= distilled water;
H2 O AUX AUX
= distilled = auxins;
water;
= auxins; BRAAUXBRA = ho-
= auxins;
= ho-
mobrassinolide;
KEL = KEL
BRA = homobrassinolide;
mobrassinolide; Kelpak KEL;==
= Kelpak
®; PHY
® PHY = Phylgreen.
Kelpak® ; PHY = Phylgreen.
Phylgreen.

Figure 2. Images of cuttings 16 days after the start of treatments. Orange arrows indicate the
callus formation.
Horticulturae 2022, 8, x FOR PEER REVIEW 5 of 9
Horticulturae 2022, 8, x FOR PEER REVIEW 5 of 9

Horticulturae 2022, 8, 561 Figure 2. Images of cuttings 16 days after the start of treatments. Orange arrows indicate the callus
5 of 9
Figure 2. Images of cuttings 16 days after the start of treatments. Orange arrows indicate the callus
formation.
formation.

H2O AUX BRA KEL PHY

HO2 AUX BRA KEL PHY
7
7 P = ns PP == ns
P = ns ns
6
6

(cm)
lenght(cm)
5
5
Sproutlenght 4
4
3
3
Sprout

2
2
1
1
0
0
cv. ® ®
cv. 'Michelangelo
'Michelangelo®'' cv.
cv. 'Cosmos
'Cosmos®''
Cultivar ×× Treatment
Cultivar Treatment == ns
ns
Figure
Figure3. 3.
Sprout length
Sprout of cv.
length of ‘Michelangelo
cv. ‘Michelangelo ® ’‘Cosmos
®’ and
and ‘Cosmos ® ’ cuttings,
®’ cuttings, 14 weeks
14 after
weeks theafter
treatments.
the treat-
Figure 3. Sprout length of cv. ‘Michelangelo®’ and ‘Cosmos®’ cuttings, 14 weeks after the treatments.
Cuttings
ments. were treated
Cuttings werewith: H2O
treated = distilled
with: H 2 O = water;
distilledAUX =
water;auxins;
AUX = BRA = homobrassinolide;
auxins; BRA = KEL =
homobrassinolide;
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,
p-values
Kelpak ; PHY = Phylgreen. Two-way and one-way ANOVA p-values are reported in the figurein(ns,
KEL =® Kelpak ; PHY = Phylgreen. Two-way and one-way ANOVA are reported the
not significant).
figure
not (ns, not significant).
significant).

Figure 4. Root fresh weight (A), dry weight (B), length (C), and area (D) of cv. ‘Michelangelo ® ’ and
Figure 4. Root
® fresh weight (A), dry weight (B), length (C), and area (D) of cv. ‘Michelangelo®®’ and
Figure 4. Root fresh weight (A), dry weight (B), length (C), and area (D) of cv. ‘Michelangelo
‘Cosmos ’ cuttings, after 14 weeks from the treatments. Cuttings were treated with: H2 O = distilled ’ and
‘Cosmos®®’ cuttings, after 14 weeks from the treatments. Cuttings were treated with: H2O = distilled
‘Cosmos
water;AUX ’ cuttings,
AUX= =auxins; after
auxins;BRA 14 weeks from the
BRA= =homobrassinolide; treatments.
homobrassinolide;KEL Cuttings
KEL= =Kelpak
Kelpak ®were treated
; PHY= = with: H O
Phylgreen.Two-way= distilled
Two-wayand
2 and
water; ®; PHY Phylgreen.
water; AUX
one-way = auxins;
ANOVA BRA =and
p-values homobrassinolide;
Tukey’s post hoc KEL = Kelpak®are
comparisons ; PHY = Phylgreen.
reported in the Two-way
figure (* p < and
0.05,
one-way ANOVA p-values and Tukey’s post hoc comparisons are reported in the figure (* p < 0.05,
one-way ANOVA p-values and Tukey’s post hoc comparisons are reported in the figure (* p < 0.05,
****pp<<0.01;
0.01; *** pp<<0.001;
0.001;ns,ns,
notnot
significant). Letters
significant). in thein
Letters figure indicateindicate
the figure the levelthe
of difference significance.
level of difference
** p < 0.01; *** p < 0.001; ns, not significant). Letters in the figure indicate the level of difference
significance.
significance.
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
Horticulturae 2022, 8, 561 6 of 9

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® ’.

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:
H2 O = 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

H2 O AUX BRA KEL PHY T C×T
M 18.0 ± 1.27 − − − − − −
0
C 20.8 ± 1.79 − − − − − −
M 8.0 ± 0.95 7.2 ± 1.79 10.0 ± 5.60 8.8 ± 2.19 7.9 ± 3.77 ns
4 ns
C 12.3 ± 2.00 14.3 ± 2.71 10.7 ± 1.23 12.6 ± 1.10 14.8 ± 4.00 ns
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 *
16 ***
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 **

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 cut-
tings [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.
Horticulturae 2022, 8, 561 7 of 9

Biostimulants like seaweed extracts seem to be a promising alternative to synthetic

products in horticulture [14]. Seaweed extracts have been successfully used for the promo-
tion 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 applica-
tion. 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.
Funding: This research was funded by the Italian Ministry of Agriculture, Food and Forestry Policies,
sub-project ‘Tecnologie digitali integrate per il rafforzamento sostenibile di produzioni e trasfor-
mazioni agroalimentari (AgroFiliere)’, AgriDigit program (DM 36503.7305.2018 of 20 December 2018).
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.
Horticulturae 2022, 8, 561 8 of 9

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.

References
1. Cardoso, J.C.; Vendrame, W.A. Innovation in propagation and cultivation of ornamental plants. Horticulturae 2022, 8, 229.
[CrossRef]
2. Kentelky, E.; Jucan, D.; Cantor, M.; Szekely-Varga, Z. Efficacy of different concentrations of NAA on selected ornamental woody
shrubs cuttings. Horticulturae 2021, 7, 464. [CrossRef]
3. Akhtar, M.S.; Khan, M.A.; Riaz, A.; Younis, A. Response of different rose species to different root promoting hormones. Pak. J.
Agric. Sci. 2002, 39, 297–299.
4. Nguyen, T.H.N.; Tänzer, S.; Rudeck, J.; Winkelmann, T.; Debener, T. Genetic analysis of adventitious root formation in vivo and
in vitro in a diversity panel of roses. Sci. Hortic. 2020, 266, 109277. [CrossRef]
5. Zhang, W.; Fan, J.; Tan, Q.; Zhao, M.; Cao, F. Mechanisms underlying the regulation of root formation in Malus hupehensis stem
cuttings by using exogenous hormones. J. Plant Growth Regul. 2017, 36, 174–185. [CrossRef]
6. Parad̄iković, N.; Teklić, T.; Zeljković, S.; Lisjak, M.; Špoljarević, M. Biostimulants research in some horticultural plant species—A
review. Food Energy Secur. 2019, 8, e00162. [CrossRef]
7. Blythe, E.K.; Sibley, J.L.; Tilt, K.M.; Ruter, J.M. Methods of auxin application in cutting propagation: A review of 70 years of
scientific discovery and commercial practice. J. Environ. Hortic. 2007, 25, 166–185. [CrossRef]
8. Betti, C.; Della Rovere, F.; Piacentini, D.; Fattorini, L.; Falasca, G.; Altamura, M.M. Jasmonates, ethylene and brassinosteroids
control adventitious and lateral rooting as stress avoidance responses to heavy metals and metalloids. Biomolecules 2021, 11, 77.
[CrossRef]
9. Vardhini, B.V. Modifications of morphological and anatomical characteristics of plants by application of brassinosteroids under
various abiotic stress conditions—A review. Plant Gene 2017, 11, 70–89. [CrossRef]
10. Pacholczak, A.; Zajaczkowska,
˛ M.; Nowakowska, K. The Effect of brassinosteroids on rooting of stem cuttings in two barberry
(Berberis thunbergii L.) cultivars. Agronomy 2021, 11, 699. [CrossRef]
11. Moreno-Castillo, E.; Ramírez-Echemendía, D.P.; Hernández-Campoalegre, G.; Mesa-Tejeda, D.; Coll-Manchado, F.; Coll-García, Y.
In silico identification of new potentially active brassinosteroid analogues. Steroids 2018, 138, 35–42. [CrossRef] [PubMed]
12. Xu, L.; Geelen, D. Developing biostimulants from agro-food and industrial by-products. Front. Plant Sci. 2018, 9, 1567. [CrossRef]
[PubMed]
13. Brzozowski, L.; Mazourek, M.A. Sustainable agricultural future relies on the transition to organic agroecological pest management.
Sustainability 2018, 10, 2023. [CrossRef]
14. Baltazar, M.; Correia, S.; Guinan, K.J.; Sujeeth, N.; Bragança, R.; Gonçalves, B. Recent advances in the molecular effects of
biostimulants in plants: An overview. Biomolecules 2021, 11, 1096. [CrossRef]
15. Dmytryk, A.; Chojnacka, K. Algae as fertilizers, biostimulants, and regulators of plant growth. In Algae biomass: Characteristics and
Applications; Chojnacka, K., Wieczorek, P.P., Schroeder, G., Michalak, I., Eds.; Springer: Cham, Switzerland, 2018; pp. 115–122.
16. Patel, J.S.; Mukherjee, A. Seaweed and associated products: Natural biostimulant for improvement of plant health. In Emerging
Trends in Plant Pathology; Singh, K.P., Jahagirdar, S., Sarma, B.K., Eds.; Springer: Singapore, 2021; pp. 317–330.
17. Ali, O.; Ramsubhag, A.; Jayaraman, J. Biostimulant properties of seaweed extracts in plants: Implications towards sustainable
crop production. Plants 2021, 10, 531. [CrossRef]
18. Kisvarga, S.; Farkas, D.; Boronkay, G.; Neményi, A.; Orlóci, L. Effects of biostimulants in horticulture, with emphasis on
ornamental plant production. Agronomy 2022, 12, 1043. [CrossRef]
19. Otiende, M.A.; Nyabundi, J.O.; Ngamau, K.; Opala, P. Effects of cutting position of rose rootstock cultivars on rooting and its
relationship with mineral nutrient content and endogenous carbohydrates. Sci. Hortic. 2017, 225, 204–212. [CrossRef]
20. Vazquez-Iglesias, I.; Ochoa-Corona, F.M.; Tang, J.; Robinson, R.; Clover, G.R.; Fox, A.; Boonham, N. Facing Rose rosette virus: A
risk to European rose cultivation. Plant Pathol. 2020, 69, 1603–1617. [CrossRef]
21. Al-Saqri, F.; Alderson, P.G. Effects of IBA, cutting type and rooting media on rooting of Rosa centifolia. J. Hortic. Sci. 1996, 71,
729–737. [CrossRef]
22. Pati, P.K.; Prakash, O.; Sharma, M.; Sood, A.; Ahuja, P.S. Growth performance of cuttings raised from in vitro and in vivo
propagated stock plants of Rosa damascena Mill. Biol. Plant. 2004, 48, 609–611. [CrossRef]
23. Bannoud, F.; Bellini, C. Adventitious rooting in Populus species: Update and perspectives. Front. Plant Sci. 2021, 12, 918.
[CrossRef]
24. Farazi, E.; Afshari, H.; Hokm Abadi, H. Effect of different concentrations of brassinosteroid on physiomorphological characteristics
of five pistachio genotypes (Pistacia vera L). J. Nuts 2015, 6, 143–153.
25. Costa, J.M.; Heuvelink, E.; Van de Pol, P. Propagation by cuttings. In Reference Module in Life Sciences; Roitberg, B.D., Ed.; Elsevier:
Amsterdam, The Netherlands, 2017.
26. Shao, F.; Wang, S.; Huang, W.; Liu, Z. Effects of IBA on the rooting of branch cuttings of Chinese jujube (Zizyphus jujuba Mill.) and
changes to nutrients and endogenous hormones. J. For. Res. 2018, 29, 1557–1567. [CrossRef]
Horticulturae 2022, 8, 561 9 of 9

27. Krajnc, A.U.; Ivanus, A.; Kristl, J.; Susek, A. Seaweed extract elicits the metabolic responses in leaves and enhances growth of
Pelargonium cuttings. Eur. J. Hortic. Sci. 2012, 77, 170–181.
28. Stirk, W.A.; Tarkowská, D.; Turečová, V.; Strnad, M.; Van Staden, J. Abscisic acid, gibberellins and brassinosteroids in Kelpak® ,
a commercial seaweed extract made from Ecklonia maxima. J. Appl. Phycol. 2014, 26, 561–567. [CrossRef]
29. Aremu, A.O.; Plačková, L.; Gruz, J.; Bíba, O.; Novák, O.; Stirk, W.A.; Doleza, K.; Van Staden, J. Seaweed-derived biostimulant
(Kelpak® ) influences endogenous cytokinins and bioactive compounds in hydroponically grown Eucomis autumnalis. J. Plant
Growth Regul. 2016, 35, 151–162. [CrossRef]
30. Szabó, V.; Sárvári, A.; Hrotkó, K. Treatment of stockplants with biostimulators and their effects on cutting propagation of Prunus
marianna ‘GF 8-1’. Acta Hortic. 2011, 923, 277–282. [CrossRef]
31. Gomes, E.N.; Vieira, L.M.; Tomasi, J.D.C.; Tomazzoli, M.M.; Grunennvaldt, R.L.; Fagundes, C.D.M.; Machado, R.C.B. Brown
seaweed extract enhances rooting and roots growth on Passiflora actinia Hook stem cuttings. Ornam. Hortic. 2018, 24, 269–276.
[CrossRef]
32. Faize, M.; Faize, L.; Burgos, L.; Critchley, A.; Albuquerque, N. Application of Ascophyllum nodosum-based soluble extract on
micropropagation and regeneration of Nicotiana benthamiana and Prunus domestica. Plants 2021, 10, 1354. [CrossRef]
33. Fleming, T.R.; Fleming, C.C.; Levy, C.C.; Repiso, C.; Hennequart, F.; Nolasco, J.B.; Liu, F. Biostimulants enhance growth and
drought tolerance in Arabidopsis thaliana and exhibit chemical priming action. Ann. Appl. Biol. 2019, 174, 153–165. [CrossRef]
34. Majda, M.; Robert, S. The role of auxin in cell wall expansion. Int. J. Mol. Sci. 2018, 19, 951. [CrossRef] [PubMed]