Scientia Horticulturae 307 (2023) 111491

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Physiological and anatomical aspects of the rooting of Brunfelsia

pauciflora cuttings
Cassiano Emilio De Sousa *, Fabrícia Lorrane Rodrigues Oliveira , Bruno Francisco Sant’Anna-
Santos , Katia Christina Zuffellato-Ribas
Postgraduate Program in Agronomy and Plant Production, Universidade Federal do Paraná (UFPR), Rua dos Funcionários, 1540, CEP 80.035-050, Curitiba, Paraná,
Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Brunfelsia is an important genus of the Solanaceae family due to its ornamental aspects and pharmacological
Ornamental plant properties. Brunfelsia pauciflora stem cuttings represent an alternative to seedling production for its propagation.
Yesterday-today-tomorrow There is no scientific data currently available concerning B. pauciflora cuttings, although previous studies with
Manaca raintree
Brunfelsia uniflora indicated that it may be difficult to root. Anatomical barriers have been reported as limiting
Auxin
factors for some species that are difficult to root. We investigated the effects of the plant growth regulator
IBA
Rhizogenesis indolebutyric acid (IBA) and the influence of the seasons of the year on the rooting of B. pauciflora stem cuttings
Anatomical barrier and compared its rooting to B. uniflora. We also investigated the possible influence of anatomical barriers and
starch contents on their rooting. Cuttings of B. pauciflora (7 ± 1 cm long) were made in the autumn of 2019,
spring/2019, and summer/2020, and treated with IBA at concentrations of 0, 2000, 4000, and 8000 mg L− 1. For
comparison, similar cuttings of B. uniflora were prepared during spring/2019 and treated with IBA concentra­
tions of 0, 4000, and 8000 mg L− 1. The rooting variables of both species were analyzed after 120 days of growth
in a greenhouse. For the anatomical analyses, segments of the base of the cuttings were collected on the day of
their collection, and again after the 120 day growth period. The samples were embedded in historesin, sectioned,
and stained with toluidine blue and Lugol’s iodine solution. No combined influence of plant growth regulator
concentrations and the season of the year was identified, indicating that those factors act separately on the
adventitious rooting of B. pauciflora. The IBA plant growth regulator did not significantly affect any of the
variables analyzed. The greatest rooting of B. pauciflora (38.8%), the highest leaf maintenance (82.0%), and the
lowest mortality rate (11.2%) were observed in the summer. The species can be classified as difficult-to-root.
B. uniflora, on the other hand, evidenced a greater rooting capacity (52.2%) in the spring than B. pauciflora
(12.2%). The use of IBA (4000 mg L− 1) resulted in an increased rooting rate of B. uniflora. Anatomically, it was
possible to identify the origin of the root primordia as the vascular cambium region, with root formation being
defined as direct. A ring of perivascular fibers, often indicated in the literature as an anatomical barrier, was not
observed as such in B. pauciflora, as rooting not only overcame the fibers, but also the callus mass. The two
species of Brunfelsia differed in terms of their starch contents, which was greater in B. uniflora and appeared to
favor its rooting.

1. Introduction
Brunfelsia L. is a genus of the Solanaceae family that has both orna­
mental (UF, 2015) and pharmaceutical importance (Magalhães et al.,
2019). The genus is native to South America and the Caribbean, and is
widely distributed throughout Brazil (Plowman, 1998; Filipowicz and
Renner, 2012; Ribeiro-Silva and Knapp, 2020). Both Brunfelsia pauciflora
(Cham. & Schltdl.) Benth. and Brunfelsia uniflora (Pohl) D. Don. are
popularly known as “yesterday-today-tomorrow”, “lady of the night”,
and the “manaca raintree” (Filipowicz and Renner, 2012; Lorenzi,
2013), are widely employed in landscape gardening in Brazil, can be
recognized by their fragrance and the color change (violet to white) of
their flowers, and are difficult to distinguish (Plowman, 1998; Lorenzi,
2013). Brunfelsia species are also appreciated for their pharmacological

* Corresponding author.
E-mail addresses: cassiano.emilio@ufpr.br (C.E. De Sousa), brunofrancisco@ufpr.br (B.F. Sant’Anna-Santos).

https://doi.org/10.1016/j.scienta.2022.111491
Received 14 October 2021; Received in revised form 28 August 2022; Accepted 2 September 2022
Available online 12 September 2022
0304-4238/© 2022 Elsevier B.V. All rights reserved.
C.E. De Sousa et al. Scientia Horticulturae 307 (2023) 111491

Fig. 1. Stock plants used in the study [1.5 column image]. A: Shrub of B. pauciflora. B: Shrub of B. uniflora. C: B. pauciflora frost damaged shrubs, during the winte.

temperatures generally facilitate rhizogenesis (Rohde and Bhalerao,

Table 1
2007, Franzon, 2010).
Effects of seasons and IBA concentrations on the rooting of B. pauciflora. Means
Interactions between the process of protrusion of the radicle from the
followed by the same letter (a, b or c) for each effect (season or IBA concen­
tration), in the same column, are not significantly different by the Tukey test at p disturbed stem and anatomical barriers (such as a continuous ring of
< 0.05. vascular fibers) could explain the low rooting percentages often seen
(Stuepp et al., 2014; Rickli et al., 2015; Bryant and Trueman, 2015;
Rooting Number of Roots/ Root Length
cutting (cm)
Pimenta et al., 2017; Denaxa et al., 2019; Monder et al., 2019; Soares
et al., 2020). Large amounts of starch, on the other hand, may increase
Season Autumn 0% c 0c 0.00 c
rooting percentages (Lima, 2011; Oliveira and Ribeiro, 2013; Marler,
Spring 13.4% b 1.28 b 0.15 b
Summer 38.8% a 2.58 a 0.49 a 2018; Pimenta et al., 2017).
IBA 0 18.3% a 1.05 a 0.20 a We therefore examined the effects of IBA and the different seasons on
concentration 2000 16.3% a 1.19 a 0.22 a the rooting of stem cuttings of B. pauciflora, compared its rooting with
(mg L− 1) 4000 13.8% a 1.37 a 0.19 a
another species of the genus (B. uniflora), and investigated the possible
8000 21.2% a 1.55 a 0.26 a
relationships of anatomical barriers and starch contents with the rooting
of those two species by addressing the following questions: 1) Can
properties, as described in the Brazilian ethnopharmacopeia (Tribess vegetative propagation be an effective alternative for propagating
et al., 2015; Yazbek et al., 2016; Magalhães et al., 2019; Ribeiro-Silva B. pauciflora? 2) Do the seasons of the year and IBA concentrations have
and Knapp, 2020; Siqueira et al., 2020). positive influences on rhizogenesis in this species? 3) Are the effects of
Considering the ornamental and pharmacological value of Brunfelsia IBA similar in B. pauciflora and B. uniflora? 4) Are there anatomical
sp., stem cuttings can be an efficient method of reproduction. Cuttings barriers that can hinder the rooting of cuttings of the two Brunfelsia
can overcome low seed availability, anticipate flowering, and maintain species? 5) Is the presence of starch related to the rooting of the two
the stability of active compounds produced by the plants (Ferrari et al., species analyzed?
2004; Guimarães et al., 2019).
Plant growth regulators can often be employed to increase the 2. Material and methods
rooting rates of stem cuttings. Indolebutyric acid (IBA) is widely used for
that purpose because of its low phytotoxicity to a wide range of different Two experiments were conducted in this study. Experiment I was
species (Franzon, 2010; Hartmann, 2014). The different seasons of the carried out with Brunfelsia pauciflora during 3 seasons of the year, with 4
year can also influence the rooting of stem cuttings, as warmer different concentrations of IBA each, and 4 repetitions of each IBA

Fig. 2. Cuttings of B. pauciflora after 120 days of incubation under greenhouse conditions, with callus, rooted, and dead stem cuttings [1.5 column image]. A: Cutting
at the beginning of the experiment. B: Rooted cutting in the spring C: Cutting with callus (arrowhead) in autumn. Detail of callus at the base of a cutting. D: Dead
cutting. Stem cutting size: 7 cm.

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2.1. Plant material and growing conditions

In Experiment I, stem cuttings of B. pauciflora were collected from the

Horto Municipal de Curitiba, located in the city of Curitiba, Paraná
State, Brazil (Fig. 1A). In Experiment II, stem cuttings of B. uniflora were
collected from a private garden (also in Curitiba) (Fig. 1B). Both stock
plants were identified using an identification key (Soares et al., 2011).
The climate of Curitiba is classified by the Köppen-Geiger system as
type Cfb (Beck et al., 2018): temperate humid with mild summers and
winters, with frequent frosts and a tendency to concentrate rainfall in
the summer months, without a well-defined dry season. The rooting
experiments were conducted in a climate-controlled greenhouse (25 ◦ C
± 2 ◦ C, with 85% relative humidity) located in the Setor de Ciências
Biológicas (25.44º S x 49.23º W, 920 m a.s.l.) of the Universidade Fed­
eral do Paraná (UFPR) in Curitiba, Paraná State, Brazil.

Fig. 3. Effects of IBA on the percentages of B. pauciflora cuttings with: callus

(C), dead cuttings (M), and cuttings that retained their original leaves (LM) Table 3
[single column image]. Means followed by the same letter (a, b or c) are not Means of the independent variables of B. pauciflora and B. uniflora cuttings
significantly different by the Tukey test at p < 0.05. submitted to 3 different concentrations of IBA. Variables: rooted cuttings (R),
number of roots per cutting (NR), and cuttings with sprouts (S) after 120 days of
growth under greenhouse conditions. Means followed by the same letter (a, b or
c) for each effect (species or IBA concentration), in the same column, are not
significantly different by the Tukey test at p < 0.05.
R NR S
Species B. pauciflora 12.2% b 1.44 b 5.22% b
B. uniflora 52.2% a 3.91 a 50.6% a

IBA Concentration (mg L− 1) 0 25.0% b 2.22 a 19.2% b

4000 35.0% a 2.81 a 35.8% a
8000 36.7% a 3.01 a 28.7% ab

Fig. 4. Effects of the different seasons on the percentage of B. pauciflora cut­

tings with callus (C), dead cuttings (M), and cuttings that retained their original
leaves (LM) [single column image]. Means followed by the same letter (a, b or
c) are not significantly different by the Tukey test at p < 0.05.

treatment; Experiment II was carried out with Brunfelsia uniflora in the

spring, with 3 different concentrations of IBA each, and 3 repetitions for
each concentration tested. The results were compared with those ob­
tained with B. pauciflora in Experiment I. Statistical analyses were
employed to assess the variances. Anatomical analyses were performed
with both species.
Fig. 5. Comparisons of cuttings [single column image]. A: B. uniflora.
B: B. pauciflora.

Table 2
Analysis of variance (average squares) of the stem cuttings of B. uniflora and B. pauciflora exposed to different concentrations of indolebutyric acid (IBA) after 120 days
of growth in a greenhouse, in Curitiba, Brazil, for the following variables: rooted cuttings (R), number of roots per cuttings (NR), average root length/cutting (RL), dead
cuttings (M), cuttings with callus (C), cuttings with sprouts (S) and maintenance rate of original leaves (LM). * Significant (P ≤ 0.05); ** Significant (P ≤ 0.01). ns: not
significant.
Biometric data
Source of variation GL R NR RL M C S LM

Species (S) 1 7200.00** 27.54** 24.33** 624.22 ns 3389.38** 9248** 5760.22**

IBA Concentration (T) 2 238.89* 1.025 ns 2.21** 430.05 ns 1165.72** 419.39 ns 694.06*
ExT 2 116.67 ns 0.659 ns 19.01** 1333.4** 2229.06** 327.17 ns 1612.39**
Treatments 5 1582.20** 6.18** 61.11** 830.22* 2035.79** 2148.22** 2074.62**
Error 12 58.33 0.90 0.11 170.89 123.72 110.39 136.50
Total 17
Coefficient of variation 9.9% 25.4% 22.9% 14.1% 30.7% 20.0% 27.3%
Bartlett’s test (χ2) 8.28 3.05 7.58 11.80 9.49 14.23 4.75

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Table 4 2.4. Plant growth regulator treatments – Experiment II

Comparisons of the means of B. uniflora and B. pauciflora submitted to three
different concentrations of IBA. Variables: root length (in centimeters) (RL), Cuttings of B. uniflora were collected in spring/2019. After dis­
percentage of cuttings with callus (C), maintenance of original leaves (LM), and infecting the bases of the cuttings, they were immersed for 10 s in a 50%
dead cuttings (M), with 120 days of conduction in the plant house. Capital letters hydroalcoholic solution containing 0 mg L− 1, 4000 mg L− 1, or 8000 mg
(A or B) indicate comparison in the same line. Lowercase letters (a or b) indicate
L− 1 of indolebutyric acid (IBA)
comparison in the same column. Means followed by the same letter are not
significantly different by the Tukey test at p < 0.05.
IBA Concentration (IBA) (mg L− 1) 2.5. Growth variable data collection
0 4000 8000

Root length (cm) B. pauciflora 0.24 bA 0.31 bA 0.26 bA

The following variables were evaluated in each experiment after 120
B. uniflora 1.23 aB 3.18 aA 3.38 aA days of growth under greenhouse conditions: rooting percentage (R%)
Cuttings with callus B. pauciflora 43.3% aA 46.7% aA 60.0% aA (percentage of cuttings that emitted roots at least 1 mm long), the
B. uniflora 60.0% aA 2.3% bB 5.3% bB number of roots per cutting (NR), the average length of the three longest
Leaf maintenance B. pauciflora 50.0% aA 55.0% aA 65.0% aA
roots per cutting (cm) (RL), percentage of dead cuttings (cuttings with
B. uniflora 51.7% aA 5.3% bB 5.7% bB
Dead cuttings B. pauciflora 46.7% aA 41.7% aA 25.0% aA necrotic tissue) (M%); percentage of cuttings with callus (cuttings alive,
B. uniflora 1.3% bB 40.0% aA 36.7% aA without roots, with the formation of undifferentiated cell masses at their
bases) (C%), percentage of living cuttings (cuttings without callus or
roots) (A%); percentage of cuttings with shoots (live cuttings with shoots
2.2. Preparation of the stem cuttings of new leaves) (Sh%), and percentage of cuttings that retained their
original leaves (LM).
The cuttings in both experiments were 7 cm ± 1 cm long, with a
bevel cut at the base and straight cut at the top, while maintaining a pair
of halved leaves in the apical portion. The cuttings were disinfected by 2.6. Stem anatomical analyses
immersion in sodium hypochlorite with 2% active chlorine for 10 min,
followed by rinsing in running water for 5 min (Wendling and Brondani, For both experiments (in each season of the year when the vegetative
2015). The cuttings were planted in polypropylene tubes (53 cm3) filled propagation experiments were performed), 3 samples were collected 1
with fine-grained vermiculite as the substrate. cm from the base of the cuttings on the day they were harvested, and 6
samples were collected at the end of the 120 day growth period (3
2.3. Plant growth regulator treatments – Experiment I samples of rooted cuttings, and 3 samples of cuttings with callus only
[without roots]). The samples were fixed in 70% FAA (formaldehyde,
Cuttings of B. pauciflora were collected in autumn/2019, spring/ glacial acetic acid, 70% ethyl alcohol, 1:1:18 v/v) for 24 h (Johansen,
2019, and summer/2020. It was not possible to collect cuttings during 1940) and subsequently stored in 70% ethanol. The material was then
the winters of 2019 and 2020 due to frost damage to the donor plants dehydrated in an increasing ethanol series and pre-infiltrated with a
(Fig. 1C). After disinfecting, the bases of the cuttings, they were historesin alcohol solution (1:1) for 24 h. The material was then infil­
immersed for 10 s in 50% hydroalcoholic solutions containing 0 mg L− 1, trated for 5 days in pure historesin and embedded. The embedded blocks
2000 mg L− 1, 4000 mg L− 1, or 8000 mg L− 1 of indolebutyric acid (IBA). were sectioned (10 μm) using a rotatory microtome (Olympus® CUT
4055). After staining with toluidine blue pH 4.0 (O’Brien and Mccully,
1981), permanent slides were mounted using verniz vitral® (Paiva et al.,
2006).
A histochemical test using Lugol’s iodine solution was performed in

Fig. 6. Anatomical structure (transverse section) of the base of B. pauciflora cuttings at the beginning of the experimental period [2 columns image]. A: General
aspect. B: Detail of the cortex, collenchyma and fibers. C: Detail of cortex fibers and druses. Abbreviations: col: collenchyma; dru: druse; epi: epidermis; fib: fibers;
per: peridermis; phl: phloem; pit: pith; xyl: xylem.

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C.E. De Sousa et al.
Fig. 7. Anatomical comparisons (transverse section) of the bases of B. pauciflora stem cutting at the initial and then final evaluations of the experiment, in different
seasons [1,5 column image]. A, C, E: Initial status. B, D, F: Final status; A, B: Autumn C, D: Spring E, F: Summer. Abbreviations: fib: fibers; xyl: xylem.
both experiments in the spring to detect the presence of starch in the
cuttings (Johansen, 1940).
Images were collected using a light microscope (Zeiss®) with an
attached digital camera (Sony Cyber-shot P72) in the Laboratório de
Anatomia e Biomecânica Vegetal of the Departamento de Botânica of the
Universidade Federal do Paraná (UFPR) in Curitiba, Brazil.
3. Trial design and statistical analysis
3.1. Experiment I
The experiments were performed using a 3 × 4 factorial design (3
seasons of the year x 4 concentrations of IBA), totaling 12 treatments,
with 4 repetitions of each IBA treatment. Twenty cuttings were used in
each experimental unit, totaling 960 cuttings, in an entirely randomized
design. The results were tested for homogeneity using Bartlett’s test (P
< 0.05). The variables were subsequently subjected to analysis of vari­
ance (ANOVA), and those with significant differences in relation to the F
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Scientia Horticulturae 307 (2023) 111491
test had their means compared using the Tukey test (P < 0.05 and P <
0.01).
3.2. Experiment II
The experimental design was entirely randomized, with three repli­
cates for each IBA treatment, with 20 cuttings in each experimental unit.
The means obtained were then compared to the means of the same
season (spring) of Experiment I. To compare them faithfully, the fourth
repetition of Experiment I was excluded from the analysis (three repe­
titions therefore being used in both experiments).
The experiments were then organized in a 2 × 3 factorial design (2
species x 3 IBA concentrations), with three repetitions for each con­
centration tested. The results were tested for homogeneity using Bar­
tlett’s test (P < 0.05). The variables were subsequently subjected to
analysis of variance (ANOVA) and the means with significant differences
in relation to the F test were compared using the Tukey test (P < 0.05
and P < 0.01).
C.E. De Sousa et al. Scientia Horticulturae 307 (2023) 111491

Fig. 8. Aspects (transverse section) of the callus formed by B. pauciflora cuttings after 120 days of incubation under greenhouse conditions [1,5 column image]. A:
General aspect of the callus and its location on the cuttings. B: Root rupturing the callus mass. The red border in both images indicates the callus mass. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 9. Anatomical evaluations (transverse sections) of rhizogenesis in B. pauciflora during the spring (control cutting) [1,5 column image]. A: A differentiating cell
mass (marked with *) adjacent to the cambium region (arrow). B, C, D: Protrusion of the adventitious root (arrow).

4. Results and discussion
4.1. Effects of plant growth regulators and seasons on Brunfelsia
pauciflora cuttings
The IBA concentration and seasons evidenced no significant in­
teractions (P < 0.01) in terms of any of the variables evaluated (Table 1).
There was no combined influence of IBA concentrations and season of
the year, indicating that those factors act separately in the adventitious
rooting of B. pauciflora (Table 1). The variables “live cuttings” and
“sprouted cuttings” were not analyzed here, as live cuttings were only
observed in the autumn, and were therefore a source of significant
variation. Sprouted cuttings, as a variable, showed a very high coeffi­
cient of variation, (even after transformation) and therefore was not
analyzed.
The application of IBA did not significantly affect any of the variables
(rooting, number of roots/cutting and root length) analyzed (Table 1;
Fig. 2B) thus, under the conditions used, the use of the plant growth
regulator IBA is dispensable. Even after the relatively long period of
permanence of the cuttings in the greenhouse (120 days), the rooting
percentages were relatively low (Table 1). The summer season was best
for rooting (38.8%), followed by spring (13.4%) (Table 1; Fig. 2B). Only
one of the 320 cuttings rooted in autumn. This cutting was excluded
from the statistical analysis as it represented a very large source of
variability. Rooting in autumn was therefore considered to be 0%
(Table 1; Fig. 2C).
The application of IBA did not significantly affect any of the variables
(callus, dead cuttings and cuttings that retained their original leaves)
analyzed (Fig. 3). The numbers of roots and average root lengths differed
statistically in the different seasons, being higher in summer for both
variables (Fig. 4). As highlighted by other authors (Paulus et al., 2016;
Barazetti et al., 2020), a larger number of roots per cutting implies an
increase in the substrate volume penetrated by them, with increased
water and nutrient absorption. Warmer seasons, such as spring and

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C.E. De Sousa et al.
Fig. 10. Anatomical comparisons (transverse sections) of B. uniflora and B. pauciflora cuttings in the summer, before initiating the vegetative propagation experi­
ments [1,5 column image]. A: B. uniflora. B: B. pauciflora. Abbreviations: col: collenchyma; cor: cortex; dru: druse; epi: epidermis; fib: fibers; phl: phloem; pit: pith;
xyl: xylem.
summer, are therefore more indicated for producing B. pauciflora
cuttings.
The average number of cuttings with callus (Fig. 2C) was not influ­
enced by the seasons (Fig. 4), with about 50% of the cuttings evidencing
that condition. This variable refers to cuttings that developed a mass of
undifferentiated cells at the base, but did not develop any roots (Fig. 2C).
The presence of callus has been linked to poor rooting percentages in a
number of species (Denaxa et al., 2019; Monder et al., 2019).
Cuttings maintaining the original leaves were statistically more
frequent in the summer (82.2%) as compared to the spring (52.5%) or
autumn (24.7%) (Fig. 4). Other authors had previously reported the
relationship between the maintenance of original leaves on cuttings and
their successful rooting, as they act as natural endogenous sources of
auxin (Hartmann et al., 2014; Maggioni et al., 2019; Latoh et al., 2019;
Fragoso et al., 2020).
Cuttings made in the summer had the lowest mortality rate (11.25%)
(Fig. 4). Cuttings made in the spring and autumn had statistically similar
mortality rates (37.19% and 44.38% respectively), although statistically
greater than those of the summer (Fig. 4). The high mortality rate in
autumn can be explained by the low availability of energy reserves held
in the stock plants at that time of the year, when the metabolism of
perennial plants is lowest (Rohde and Bhalerao, 2007).
4.2. Comparison of rooting of Brunfelsia pauciflora and Brunfelsia
uniflora stem cuttings
The interactions of the rooting, number of roots per cutting, and
sprouted cuttings were not significant by the analysis of variance
(ANOVA) (Table 2). Those variables were therefore evaluated separately
as they are independent variables (Table 3). The variables of average
root length/cutting, dead cuttings, cuttings with callus, and mainte­
nance rate of the original leaves, were not independent, indicating that
they may act in combined fashions (Table 2).
The variables evaluated (rooted cuttings, number of roots per cutting
and cuttings with sprouts) were statistically greater in B. uniflora than in
B. pauciflora (Table 3; Fig. 5A, B). Additionally, the application of IBA
provided higher rate of rooted cuttings in the former species as
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Scientia Horticulturae 307 (2023) 111491
compared to the control, and positively influenced its sprouting
(Table 3). The number of roots per cutting was not influenced by the
application of IBA in either species (Table 3). Both species had low
rooting rates as compared to other native Brazilian species (Lusa and
Biasi, 2011; Rossa et al., 2019; Saudade et al., 2021) and other species of
the Solanaceae family (Blum et al., 2013; Oliveira et al., 2015), although
B. uniflora showed a better rooting response than B. pauciflora.
Brunfelsia uniflora generated more roots per cuttings than
B. pauciflora (Table 3; Fig. 5A and B), which is a relevant factor for the
commercial production of seedlings, as well-developed root systems can
increase survival rates in the field (Rezende et al., 2013).
The higher percentage of sprouted cuttings of B. uniflora reflects the
greater vigor of that species as compared to B. pauciflora (Table 3;
Fig. 5A and B), as vigorous cuttings with greater energy reserves can
produce higher numbers of sprouts (Cunha et al., 2015).
The following variables were found to show significant interactions
(P < 0.01): average root length, dead cuttings, cuttings with callus, and
the maintenance of original leaves (Table 2). Those interactions were
therefore analyzed individually (Table 4). It was not possible to observe
any influence of IBA on the rooting of B. pauciflora in any of the in­
teractions analyzed, supporting the results obtained in Experiment I,
where the species did not respond to IBA applications (Fig. 3).
The number of roots on stem cuttings is linked to the production of
better plants (Bernardo et al., 2020). A well-developed root system al­
lows greater exploitation of the substrate and greater absorption of the
water and nutrients necessary for cutting establishment in the field
(Almeida et al., 2017). The application of IBA produced a positive effect
on the average root length of B. uniflora; that effect was statistically
similar with both concentrations of IBA tested (Table 4).
The rooting rate (rooted cuttings) of B. uniflora was quite high
(52.2%) (Table 3) as compared to other studies undertaken with that
same species: Oliveira et al. (2009) did not report any rooting at all,
while Althaus-Ottmann et al. (2006) reported a total of 41.9% rooted
cuttings. Such differences in the rooting rate can reflect factors such as
genotype, the mineral nutrition of the donor plant, the physiological age
of the donor plant, the type of plant material collected, and the presence
of rooting inhibitors (Hartmann et al., 2014).
C.E. De Sousa et al. Scientia Horticulturae 307 (2023) 111491

Fig. 11. Anatomical comparison (transverse sections) of cuttings stained with toluidine blue and lugol [2 columns image]. A, C: B. uniflora; B, D: B. pauciflora. A, B:
At the initiation of the experiments; C, D: At the termination of the experiments. Arrows indicate starch granules (stained black) in the cortex and pith. Abbreviation:
pra: parenchymatic rays.

Although the rooting rates of the two species examined were posi­
tively affected by the use of IBA, B. uniflora had a high mortality per­
centage (Table 4). Without the use of the plant growth regulator, the
mortality percentage of B. uniflora was lower than that of B. pauciflora
(Table 4). Even though they belong to the same genus and have very
similar morphoanatomies, B. uniflora and B. pauciflora showed different
physiological responses to IBA.
4.3. Anatomical structure of Brunfelsia pauciflora cuttings
The bases of Brunfelsia pauciflora stem cuttings, in transverse section,
are composed (externally to internally) of the following tissues:
epidermis and/or periderm (Fig. 9A), cortex with collenchyma, and
parenchyma cells (Fig. 6B) with the presence of fibers and druse crystals
(Fig. 6C). Although secondary xylem and phloem are present (Fig. 6A
and B), the pith can still be observed (Fig. 6A).
The segments that were first collected from the bases of the cuttings
at the beginning of the experiment in autumn (Fig. 7A and B), spring
(Fig. 7C and D), and summer of 2020 (Fig. 7E and F) had similar stem
anatomies. Roots were observed during spring/2019 and summer/2020,
with the absence of roots in autumn/2019.
Callus was observed on the cuttings after 120 days of incubation
under greenhouse conditions (Fig. 8A), mostly in the cortex region.
Anatomically, callus is an undifferentiated mass of parenchymatic cells

8
C.E. De Sousa et al.
(Hartmann et al., 2014). Despite the common presence of callus in
B. pauciflora, that mass was not a barrier to rooting (Fig. 8B), unlike the
situation observed with Rosa helenae (Rosaceae) "Semiplena", where the
use of plant growth regulators resulted in accumulations of callus that
obstructed root initiation and decreased its rooting percentage (Monder
et al., 2019).
Callus is a required condition for rooting in some species, although
the anatomical analysis of rooting in B. pauciflora can be classified as
direct (Fig. 9A-D). In direct rooting, there are specialized cells near the
vascular region responsible for the cell divisions that form the root
primordia (Hartmann et al., 2014). Although that characteristic is
generally associated with easy-to-root species (Hartmann et al., 2014),
that apparently is not the case with B. pauciflora.
The debate concerning possible anatomical barriers that could
hinder adventitious root intrusion is not recent (Lovell and White,
1986), and there is still no consensus in the scientific literature. The low
rooting percentages of Corymbia sp. and Eucalyptus sp. cuttings have
been associated with a sclerenchymatous ring surrounding the vascular
cylinder (Bryant and Trueman, 2015). The rooting rate of Castanea
henryi was found to be closely related to the structure of the phloem fiber
ring (Chen et al., 2020), and Amissah et al. (2008) noted that a cohesive
fiber network can restrict the expansion of cells forming root primordia.
Denaxa et al. (2019) found that to be the case with Olea europaea, a
difficult-to-root species, where the action of a plant growth regulator
was evidenced in areas external to the vascular bundle – and not at the
site of action (the cambium region). Those authors found that anatom­
ical barriers prevented the plant growth regulator from reaching the
areas of root formation, and thus hindered rooting.
Studies with Coffea arabica (Jesus et al., 2010), Paulownia fortunei
(Stuepp et al., 2014), Vochysia bifalcata (Rickli et al., 2015), Annona
crassiflora (Pimenta et al., 2017), and Hancornia speciosa (Soares et al.,
2020), however, did not associate fiber bundles with low rooting rates of
cuttings.
The fiber bundles of Brunfelsia pauciflora were not preventing root
protrusion, as growing roots were observed crossing that anatomical
structure. It is therefore likely that root formation did not occur in those
cuttings due to the lack of stimulation of the cambium cells. In cuttings
where stimulation was sufficient, there was root initiation, differentia­
tion, and vascular connections – culminating in root formation (Fig. 9)
(Hartmann et al., 2014). We suggest that a low presence of rooting co­
factors was responsible for this weak stimulus, resulting in low rooting
percentages in B. pauciflora.
4.4. Histological starch location in Brunfelsia pauciflora cuttings as
compared with Brunfelsia uniflora
Transverse sections of the bases of B. uniflora stem cutting show
(externally to internally) the following tissues: epidermis or periderm,
collenchyma, parenchyma, perivascular fibers, secondary phloem, sec­
ondary xylem, primary xylem, and pith, as was also observed in
B. pauciflora (Fig. 10A, B); both species have a band of perivascular fi­
bers in the innermost portion of the cortex (Fig. 10A, B). Rhizogenesis in
B. uniflora can be defined as direct, as was also observed in B. pauciflora;
the root primordia were found to be associated with the vascular cam­
bium region.
Based on our study, it is possible to refute the hypothesis that only
anatomical barriers are responsible for the low rooting in B. pauciflora,
as both species have very similar anatomical structures although
B. uniflora had a considerably higher rooting percentage than
B. pauciflora.
Staining with Lugol’s iodine solution evidenced greater quantities of
starch in Brunfelsia uniflora (Fig. 11A and C) than in B. pauciflora
(Fig. 11B and D) and, according to Oliveira and Ribeiro (2013), greater
starch reserves favor cutting development.
Starch is a high molecular weight polysaccharide formed by glucose
monomers; it acts as an energy reserve in plants as enzymes such as
9
Scientia Horticulturae 307 (2023) 111491
amylases and phosphorylases can cleave the starch bonds and produce
soluble sugars (Taiz et al., 2017). The presence of sugars available to the
cells that will form the root will be a determining factor for the suc­
cessful rooting of cuttings – especially during the rhizogenesis phase
when photosynthetic activity is low (Monder and Pacholczak, 2018).
In the present study, more starch granules were visible in B. uniflora
cuttings than in B. pauciflora cuttings, and those reserves may be asso­
ciated with the higher rooting percentage of B. uniflora. The higher
rooting percentages of Cycas micronesica (Marler, 2018) and Euplassa
inaequalis (Oliveira and Ribeiro, 2013) cuttings were attributed to the
presence of starch, while the low rooting observed in Annona crassiflora
cuttings (Pimenta et al., 2017) was attributed, on the other hand, to their
low starch contents.
The different starch contents observed in the present study may not
be a fixed condition of the species, however, and may vary among donor
plants and genotypes of the same species (Oliveira et al., 2012). It will
therefore be important to conduct additional studies with several
matrices of both species and evaluate if the observed differences in
starch content represents a species-specific condition. Large variations
have been reported in terms of the rooting of cuttings of different species
– depending on the cultivars elected, the age of the donor plant, and the
nature of the detached organ (Lovell and White, 1984).
Good conditions for the growth of the donor plants, in terms of
incident light, water, and nutrients will result in a higher photosynthetic
rate and their better assimilation of available nutrients (Hartmann et al.,
2014), which would result in greater starch reserves in the cuttings and
their greater rooting.
5. Conclusions
Cuttings offer a viable alternative for the vegetative propagation of
B. pauciflora, although the results of our experiments indicated it to be a
difficult-to-root species. The application of IBA did not positively in­
fluence the rooting of this species, although the summer is indicated as
the most promising season for further studies.
In contrast to B. pauciflora, B. uniflora responded to the application of
IBA. That plant growth regulator positively influenced rooting, root
length, and the percentage of B. uniflora sprouted cuttings. The indicated
IBA concentration is 4000 mgL− 1.
Both species evidenced direct rooting. The limited rooting of
B. pauciflora cuttings is apparently not associated with an anatomical
barrier, as the perivascular fibers do not act as an obstacle to
rhizogenesis.
It was possible to conclude that the visually superior quantities of
starch in B. uniflora favored its rooting.
CRediT authorship contribution statement
Cassiano Emilio De Sousa: Conceptualization, Methodology,
Investigation, Formal analysis, Writing – original draft. Fabrícia Lor­
rane Rodrigues Oliveira: Methodology, Investigation. Bruno Fran­
cisco Sant’Anna-Santos: Conceptualization, Methodology, Validation,
Resources, Visualization, Writing – review & editing, Supervision,
Project administration. Katia Christina Zuffellato-Ribas: Conceptual­
ization, Methodology, Validation, Resources, Visualization, Writing –
review & editing, Supervision, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
Data will be made available on request.
C.E. De Sousa et al. Scientia Horticulturae 307 (2023) 111491

Acknowledgments Lusa, M.G., Biasi, L.A., 2011. Estaquia de Cuphea calophylla subsp. mesostemon (Koehne)
Lourteig (Lythraceae). Re Bras. Pl. Med. 13 (1) https://doi.org/10.1590/S1516-
05722011000100008. Botucatu.
This work was supported by the Conselho Nacional de Desenvolvi­ Magalhães, K.D.N., et al., 2019. Medicinal plants of the Caatinga, northeastern Brazil:
mento Científico e Tecnológico (CNPq - National Council for Scientific ethnopharmacopeia (1980-1990) of the late professor Francisco José de Abreu
and Technological Development), the GEPE (Grupo de Estudo e Pesquisa Matos. J. Ethnopharmacol 237, 314–353. https://doi.org/10.1016/j.
jep.2019.03.032.
em Estaquia), Laboratório de Anatomia e Biomecânica Vegetal, and Maggioni, R.A., et al., 2019. Viability of the mini-cutting technique in the production of
UFPR (Universidade Federal do Paraná). Drimys brasiliensis Miers seedlings. Floresta 50 (2), 1195–1202. https://doi.org/
10.5380/RF.V50I2.62020. Curitiba, PR.
Marler, T.E., 2018. Stem carbohydrates and adventitious root formation of Cycas
Supplementary materials micronesica following Aulacaspis yasumatsui infestation. Hortic. Sci. 53 (8),
1125–1128. https://doi.org/10.21273/HORTSCI13170-18.
Supplementary material associated with this article can be found, in Monder, M.J., et al., 2019. Anatomical structure changes in stem cuttings of rambler
roses induced with plant origin preparations. Sci. Hortic. 255, 242–254. https://doi.
the online version, at doi:10.1016/j.scienta.2022.111491. org/10.1016/j.scienta.2019.05.034.
Monder, M.J., Pacholczak, A., 2018. Preparations of plant origin enhance carbohydrate
References content in plant tissues of rooted cuttings of rambler roses: rosa beggeriana
‘Polstjärnan’ and Rosa helenae ‘Semiplena’. Acta Agric. Scand. B Soil Plant Sci. 68
(3), 189–198. https://doi.org/10.1080/09064710.2017.1378365.
Almeida, J.P.N., et al., 2017. IBA concentration and substrate effects on rooting and vigor
O’Brien, P.P., Mccully, M.E., 1981. The Study of Plants Structure Principles and Select
of woody cuttings of cajaraneira. Rev. Cienc. Agrar. 60 (1), 11–18. https://doi.org/
Methods. Termarcarphi Pty. Ltda, Melbourne, Australia.
10.4322/rca.2004 jan./mar.
Oliveira, J.A.R., et al., 2015. Tipos de estacas e uso de AIB na propagação vegetativa de
Althaus-Ottmann, M.M., et al., 2006. Propagação vegetativa de manacá (Brunfelsia
Fisális. Agro@mbiente on-line 9 (3), 342–346. https://doi.org/10.18227/1982-
uniflora (Pohl.) D. Don.). Re Bras. Hortic. Ornam. 12 (1), 31. https://doi.org/
8470ragro.v9i3.2571.
10.14295/rbho.v12i1.4, 36.
Oliveira, M.C., Ribeiro, J.F., 2013. Enraizamento de estacas de Euplassa inaequalis (Pohl)
Amissah, J.N., et al., 2008. Adventitious root formation in stem cuttings of Quercus
Engl. de mata de galeria em diferentes estações do ano. Biosci. J. 29 (4), 991–999.
bicolor and Quercus macrocarpa and its relationship to stem anatomy. J. Am. Soc.
Uberlândia.
Hortic. Sci 133 (4), 479–486. https://doi.org/10.21273/JASHS.133.4.479.
Oliveira, R.J.P., et al., 2012. Teores de carboidratos em estacas lenhosas de mirtileiro. Re
Barazetti, M., et al., 2020. Cocoa shell substrate, indolebutyric acid doses and length of
Bras. Frutic. 34 (4), 1199–1207. https://doi.org/10.1590/S0100-
cuttings in the rooting of Fridericia chica. Pesq. Agropec. Tropical. Goiânia 50,
29452012000400029. Jaboticabal.
e60122. https://doi.org/10.1590/1983-40632020v5060122.
Paiva, J.G.A., et al., 2006. Verniz vitral incolor 500®: uma alternativa de meio de
Beck, H.E., et al., 2018. Present and future Köppen-Geiger climate classification maps at
montagem economicamente viável. Acta Bot. Bras. 20 (2), 257–264. https://doi.org/
1-km resolution. Sci. Data 5, 180214. https://doi.org/10.1038/sdata.2018.214,
10.1590/S0102-33062006000200002.
10.1038/sdata.2018.214.
Paulus, D., et al., 2016. Ácido indolbutírico na propagação vegetativa de alecrim. Hortic.
Bernardo, B.E.D.C., et al., 2020. Propagação Por Estaquia De Erva-Baleeira (Cordia
Bras. 34 (4), 520–528. https://doi.org/10.1590/S0102-053620160411. Vitoria da
verbenacea). Re em Agronegócio e Meio Ambiente 13 (3), 947–957. https://doi.org/
Conquista.
10.17765/2176-9168.2020v13n3p947-957.
Pimenta, A.C., et al., 2017. Estaquia e anatomia caulinar de Annona crassiflora Mart. Cad.
Blum, C.T., et al., 2013. Propagação de Cestrum corymbosum por sementes e estaquia.
Cienc. Agrar 9 (2), 01–07.
Floresta 43 (1), 137–144. https://doi.org/10.5380/rf.v43i1.25012.
Plowman, T.C., 1998. A revision of the South American species of Brunfelsia
Bryant, P.H., Trueman, S.J., 2015. Stem anatomy and adventitious root formation in
(Solanaceae). Field Mus. Nat. Hist. 39.
cuttings of Angophora, Corymbia and Eucalyptus. Forests 6, 1227–1238. https://doi.
Rezende, F.P.F., et al., 2013. Aplicação de extratos de folhas e tubérbulos de Cyperus
org/10.3390/f6041227.
rotundus L. e de auxinas sintéticas na estaquia caulinar de Duranta repens L. Re Bras.
Chen, W., et al., 2020. Factors involved in the success of Castanea henryi stem cuttings in
Pl. Med. 15 (4), 639–645. https://doi.org/10.1590/S1516-05722013000500003.
different cutting mediums and cutting selection periods. J. For. Res. https://doi.org/
Botucatusupl. 1.
10.1007/s11676-020-01208-5.
Ribeiro-Silva, S., Knapp, S., 2020. Brunfelsia in Flora do Brasil 2020. Jardim Botânico do
Cunha, A.L.B., et al., 2015. Propagação vegetativa de estacas de Piper hispidum Sw. em
Rio de Janeiro.
diferentes substratos. Re Bras. Pl. Med., Campinas 17 (4), 685–692. https://doi.org/
Rickli, 2015. Origem de brotações epicórmicas e aplicação de ácido indolilbutírico no
10.1590/1983-084X/14_113 supl. I.
enraizamento de estacas de Vochysia bifalcata Warm. Cienc. Florest. 25 (2), 385–393.
Denaxa, N.K., et al., 2019. Assessing the effect of oxidative enzymes and stem anatomy
https://doi.org/10.5902/1980509818457. Santa Maria.
on adventitious rooting of Olea europaea (L.) leafy cuttings. Span. J. Agric. Res. 17
Rohde, A., Bhalerao, R.P., 2007. Plant dormancy in the perennial context. Trends Plant
(3), e0803. https://doi.org/10.5424/sjar/2019173-14486.
Sci. 12, 217–223. https://doi.org/10.1016/j.tplants.2007.03.012.
Ferrari, M.P., et al., 2004. Propagação Vegetativa De Espécies Florestais. Embrapa
Rossa, 2019. Cuttings of Euphorbia phosphorea Mart and Euphorbia enterophora Drake at
Florestas, Colombo, p. 19.
different concentrations of indole-butyric acid and analysis of economic viability.
Filipowicz, N., Renner, S.S, 2012. Brunfelsia (Solanaceae): a genus evenly divided
Ornam. Hortic. 25 (3), 314–323. https://doi.org/10.1590/2447-536X.v25i3.1985.
between South America and radiations on Cuba and other Antillean islands. Mol.
Campinas.
Phylogenetics Evol. 64 (1), 1–11. https://doi.org/10.1016/j.ympev.2012.02.026.
Saudade, N.S., et al., 2021. Ácido indolbutírico na estaquia de Calliandra brevipes e
Fragoso, R.O., et al., 2020. Ficus enormis cuttings rooting: concentrations of indole
Calliandra tweedii. Ad For. Sci. 8 (1), 1327–1333. https://doi.org/10.34062/afs.
butyric acid and seasonal variations. BOSQUE. Valdivia, Chile, pp. 373–379. https://
v8i1.10123. Cuiabá.
doi.org/10.4067/S0717-92002020000300373.
Siqueira, B.V.L., et al., 2020. Mercury: the beginnings in the medicalization of common
Franzon, R.C., et al., 2010. Propagação Vegetativa por Estaquia. Produção de Mudas:
names of medicinal plants in Brazil. Rodriguesia 71, e00972019. https://doi.org/
principais técnicas utilizadas na propagação de fruteiras. Embrapa Cerrados,
10.1590/2175-7860202071056.
Planaltina, pp. 37–43. https://ainfo.cnptia.embrapa.br/digital/bitstream/item/
Soares, E.L.C., et al., 2011. Taxonomic synopsis and analytical key for the genera of
77778/1/doc-283.pdf.
Solanaceae from Rio Grande do Sul, Brazil. Acta Bot. Bras. 25 (2) https://doi.org/
Guimarães, R.N., et al., 2019. Vegetative propagation of Pequi (Souari nut) by cutting.
10.1590/S0102-33062011000200011.
Cienc. Rural 49 (2). https://doi.org/10.1590/0103-8478cr20180579.
Soares, F.S., et al., 2020. Aspectos anatômicos de impedimentos ao enraizamento em
Hartmann, H.T., et al., 2014. Plant Propagation: principles and Practices. Pearson
estacas caulinares de mangabeira. Acta Iguazu 9 (1), 82–89. https://doi.org/
Education, Harlow, UK, 8ª ed.
10.48075/actaiguaz.v9i1.21687. Cascavel.
Jesus, A.M.S., et al., 2010. Observações anatômicas em plantas de Coffea arabica L.
Taiz, L., et al., 2017. Fisiologia e desenvolvimento vegetal. Artmed Editora.
obtidas por enraizamento de estacas. Re Ceres, Viçosa 57 (2), 175–180. https://doi.
Tribess, B., et al., 2015. Ethnobotanical study of plants used for therapeutic purposes in
org/10.1590/S0034-737x2010000200006.
the Atlantic Forest region, Southern Brazil. J. Ethnopharmacol. 164, 136–146.
Johansen, D.A., 1940. Plant Microtechique. McGraw-Hill Book Company, Inc., London.
https://doi.org/10.1016/j.jep.2015.02.005.
Latoh, L.P., et al., 2019. Can indolebutyric and fulvic acids induce adventitious
Yazbek, P.B., et al., 2016. Plants used during maternity, menstrual cycle and other
rhizogenesis on mini-cuttings from Brazilian native Tibouchinas? Ornam. Hortic. 25
women’s health conditions among Brazilian cultures. J. Ethnopharmacol. 179,
(1) https://doi.org/10.14295/oh.v25i1.1257. Viçosa.
310–331. https://doi.org/10.1016/j.jep.2015.12.054.
Lima, et al., 2011. Capacidade de enraizamento de estacas de Maytenus muelleri
University of Florida, 2015. Florida-Friendly LandscapingTM Plant List. The Florida
Schwacke com a aplicação de ácido indol butírico relacionada aos aspectos
Friendly LandscapingTM Guide to Plant Selection & Landscape Design. University of
anatômicos. Re Bras. Pl. Med. 13 (4), 422–438. https://doi.org/10.1590/S1516-
Florida Institute of Food and Agricultural Sciences, Gainesville, p. 52. https://ffl.ifas.
05722011000400008. Botucatu.
ufl.edu/pdf/FYN_Plant_Selection_Guide_2015.pdf.
Lorenzi, H., 2013. Plantas Para Jardim No Brasil: herbáceas, Arbustivas e Trepadeiras.
Wendling, I., Brondani, G., 2015. Vegetative rescue and cuttings propagation of
Instituto Plantarum, Nova Odessa, SP, 2a ed.
Araucaria angustifolia. Re Arvore 39 (1), 93–104. https://doi.org/10.1590/0100-
Lovell, P.H., White, J., 1986. Anatomical Changes During Adventitious Root formation.
67622015000100009.
New root Formation in Plants and Cuttings. Springer, Dordrecht, pp. 111–140.
https://doi.org/10.1007/978-94-009-4358-2_4.

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