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Folia
Horticulturae
https://sciendo.com/journal/FHORTORIGINAL ARTICLE Open access
ORIGINAL ARTICLE
Folia Hort. 35(2) (2023): 1–16
DOI: 10.2478/fhort-2023-0029 • AoP
Enhanced germination of seeds native to Brazil:
A comparative analysis between free and nanoencapsulated
gibberellic acid in Dyckia sp. (Bromeliaceae)
*Corresponding authors.
e-mail: jc_baudraz@live.com (Jean Carlo Baudraz de Paula); shimizu@uel.br (Gabriel Danilo Shimizu).
1 Agronomy Department, Universidade Estadual de Londrina (UEL), Londrina, 86051-990, Brazil
2 Environmental Nanotechnology Lab, Science and Technology Institute of Sorocaba (ICTS), São Paulo State University (UNESP),
Sorocaba, Av. Três de Março, 511 - Alto da Boa Vista, Sorocaba - SP 18087-180, Brazil
ABSTRACT
Brazil is home to a great diversity of species of the genus Dyckia. However, many of these species are threatened due to
habitat destruction and predatory exploitation. An alternative to conserving these plants is sexual propagation using plant
regulators to stimulate germination. Gibberellic acid (GA3
and ease of degradation pose challenges. Therefore, nanoencapsulation of GA3 could be used to protect the molecule
3 were evaluated on the germination of
four species: D. cabrerae, D. dusenii, D. pottiorum and D. walteriana
3, in which the species D. dusenii and D. walteriana3,
with an increase from 35% to more than 60% germination. However, the species D. cabrerae and D. pottiorum responded
positively to GA3 only in vegetative growth parameters. In the second stage, the use of nanoparticles of alginate/chitosan
(NP ALG/CS) and chitosan/tripolyphosphate (NP CS/TPP) containing GA3 was compared with free GA3 and with NPs
without GA33
D. walteriana–1–1), mainly with the ALG/
CS NPs. Therefore, the use of GA3 is recommended for D. dusenii and D. walteriana, and for the latter, nanoparticles
containing ALG/CS-GA3 allow a reduction in the required dose.
Keywords: bromeliads, domestication, gibberellins, nanotechnology, plant growth regulators
Jean Carlo Baudraz de Paula1,*, Hugo Roldi Guariz1, Kauê Alexandre Monteiro
de Moraes1, Gabriel Danilo Shimizu1,*, Ricardo Tadeu de Faria1, Halley Caixeta
de Oliveira1, Leonardo Fernandes Fraceto2, Anderson Espirito Santo Pereira2
INTRODUCTION
Bromeliaceae, with 1,712 species catalogued (Gouda
et al., 2022). Among the states with the highest
concentration of species, Bahia (358), Espírito Santo
(334), Rio de Janeiro (333) and Minas Gerais (331) stand
out, with species occurring mainly in the Atlantic Forest
of bromeliads on a commercial scale is a viable and
well-explored activity in Brazil, with highlighted use
in landscaping projects and for decoration of indoor
environments, due to the easy maintenance, hardiness
2 Free and nanoencapsulated gibberellic acid in the germination of Dyckia sp.
environments (Anacleto and Negrelle, 2013). The
increases their added value. In this sense, bromeliads of
the genus Dyckia sp. stand out for the absence of the
aesthetically original morphology (Pinangé et al., 2017).
Due to the increased use of bromeliads in Brazilian
landscaping, large numbers of plants end up being
removed from nature, which is one of the factors
contributing to the increase in endangered species
(Pereira et al., 2010). Among the Dyckia species, for
example, D. walteriana Leme. falls into the ‘critically
endangered’ category, requiring urgent measures for
the conservation of populations (Musegante et al., 2020;
Interest in the propagation of native species
available for the management and analysis of the seeds
characterise their physical and physiological attributes.
It is crucial to understand the behaviour of growth and
development factors in native species as this allows
the creation of cultivation protocols. These protocols,
addition to contributing to the recovery of already
degraded areas (Ribeiro et al., 2012; Borges et al., 2018).
For the germination process to occur uniformly and
with the highest possible percentage, some procedures
can be adopted in the seeds, such as the application
of plant growth regulators, for example, gibberellic
acid (GA3). Gibberellins promote germination through
several metabolic state changes in the seed that allow the
embryo to re-establish its activity (Hossel et al., 2018).
One way of stimulati ng germ ination provided by GA3
is through the induction of the production of hydrolytic
enzymes (such as amylases) in the endosperm, which
generating soluble organic compounds that provide
energy for the germinating embryo (Hossel et al.,
2018). There are reports of improvement in germination
and overcoming dormancy of seeds of several species
with the use of GA3 (Hossel et al., 2018). Despite this,
one of the obstacles to the application of plant growth
regulators is that they are easily degraded when exposed
to environmental factors, such as light and temperature,
resulting in the loss of their activity. In this context,
the use of nanotechnology aims to provide nanoscale
materials capable of improving stability and activity,
in addition to reducing possible environmental impacts
(Ashraf et al., 2021). Thus, nanoencapsulation provides
advantages such as longer action time, increased
biological activity, and reduced concentration required
toxic metabolites and is easily degraded, hence the
option to use natural biodegradable polymers, such as
alginate and chitosan (Francisco and García-Estepa,
2018; Pascoli et al., 2018).
Studies associating nanoparticles (NPs) containing
plant regulators such as GA3 are still in infancy and
mainly aimed at large crops such as beans (Phaseolus
vulgaris L.) (Pereira et al., 2017), cucumber (Cucumis
sativus L.) (Yang et al., 2018) and tomato (Solanum
lycopersicum var. cerasiforme) (Pereira et al., 2019).
Therefore, the application of this technology to improve
the germination of seeds with low vigour and for use in
ornamental species, such as those of the genus Dyckia,
3 in four species of Dyckia. In addition,
the potential of nanoencapsulated GA3 to stimulate
seed germination in one of the species responsive to the
growth regulator was evaluated.
MATERIALS AND METHODS
Plant material and lot characterisation
The experiment was conducted at the Seed Laboratory
of the State University of Londrina (UEL). Ripe fruits
of D. cabrerae L. B. Smith & Reitz, D. dusenii L. B.
Smith, D. pottiorum Leme., and D. walteriana Leme.
were collected from mother plants, approximately 90
of collector Dr. Walter Miguel Kranz, located in the
The fruits were allocated in Kraft® (Londrina-PR)
In a shaded place, they were dried in the open air for 3
days to facilitate the extraction of the seeds, which were
stored in Kraft® paper bags, in the refrigerator at 7.5 ±
1.0°C and at a relative humidity (RH) of 26 ± 7%, for
270 days. For the characterisation of the lot, the water
content and viability of the seeds were determined by
of the Rules for Seed Analysis (BRASIL, 2009). This
characterisation occurred after the harvest (10 days) and
before the installation of the experiment (after 270 days).
For the determination of water content, 0.2 g of seeds
of each species were placed in an oven at 105 ± 3°C for 24
hr. For carrying out the tetrazolium test, four repetitions
of 25 seeds each were used. For each species, the seeds
were placed in cryovials with a capacity of 2 mL and
subsequently completed with distilled water. After 24
hr at 25°C in a germination chamber, the water was
removed, and a 1% tetrazolium salt solution was added.
The seeds then remained in a germination chamber in
the absence of light for 24 hr at 30°C. After this period,
the percentage of viable seeds was evaluated with the
aid of a magnifying glass. To calculate the percentage of
viable seeds, empty seeds were not considered, that is,
those without an embryo.
Preparation of formulations
Gibberellic acid (GA3—90%), sodium alginate (ALG),
chitosan (CS) and tripolyphosphate (TPP) were obtained
Baudraz de Paula et al. 3
from Sigma-Aldrich® (São Paulo-SP). The NPs
were provided by Professor Dr. Leonardo Fernandes
Fraceto from Universidade Estadual Paulista (UNESP/
Sorocaba) following the methodology of Pereira et al.
(2017 ).
For the preparation of free GA3 solutions, the
product was weighed and diluted in alcohol (70%),
and then distilled water was added to obtain an initial
concentration of 50 mg L–1. Next, this solution was
diluted in distilled water to obtain the desired doses
(0.25, 0.5, 0.75, 1.0, 2.5 and 5.0 mgL–1).
The nanoformulations were prepared at an initial
concentration of 50 mg L–1. Alginate/chitosan (ALG/
CS) NPs were prepared by the ionotropic pre-gelation
method. Initially, with the aid of a peristaltic pump
(Miniplus 3, Gilson® (São Paulo-SP)), 3.75 mL of
calcium chloride (CaCl2) solution (18 mM) was added
to 59 mL of ALG solution (0.063%, pH 4.9) under
strong magnetic stirring (500 rpm). During this step,
2 on ALG occurs through ionic
interactions, forming a structure called an ‘egg box’.
Subsequently, GA3 was added under stirring until
complete dissolution to reach the desired concentration
(50 mg L–1). The ALG/CaCl2/GA3
under agitation, and 12.5 mL of an aqueous solution of
chitosan (0.07% pH 4.6), prepared in an aqueous solution
containing 5.7% of acetic acid, was added over 90 min,
forming a polyelectrolyte complex between polymers.
The same process occurred without the presence of the
GA3 regulator to obtain the ALG/CS NPs without the
growth regulator.
Chitosan/tripolyphosphate (CS/TPP) NPs were
First, 10 mL of a CS solution (0.2%, pH 4.5), prepared in
vigorous agitation (500 rpm), and GA3 was added until
L–1. After dissolution
of GA3, 6 mL of TPP solution (0.1%, pH 4.5 at 4°C) was
added. CS/TPP NPs were also prepared without the
presence of the regulator.
The formulations were characterised by size, zeta
performance liquid chromatography—HPLC), the
polydispersity index (PDI) and pH. The ALG/CS NPs
with and without GA3 showed an average size of 450 ±
10 nm, PDI of 0.3, zeta potential of –29 ± 0.5 mV, pH
NPs with and without GA3 showed an average size of
195 ± 1 nm, PDI of 0.3, zeta potential of +27 ± 3 mV,
NPs remained stable for 60 days at room temperature
(Pereira et al., 2017).
Tre a tm e n t s
In all experiments, the seeds were sterilised in a 1%
sodium hypochlorite solution (1 min), followed by
immersion in 70% alcohol (1 min) and subsequently
min at room temperature. In the control treatment (0 mg
L–1
3, seeds of D. cabrerae,
D. dusenii, D. pottiorum and D. walteriana were
submitted to doses of 0.25, 0.5, 0.75, 1.0, 2.5 and 5 mg
L–1 of GA3. In the experiment with NPs, D. walteriana
seeds were submitted to the following treatments: NPs
of ALG/CS and CS/TPP containing GA3 and NPs of
ALG/CS and CS/TPP without GA3. The doses used
were 0.25, 0.5, 0.75, 1.0, 2.5 and 5.0 mgL–1 of GA3
and the corresponding dilutions of the NPs without the
regulator. Free GA3 data in this experiment were based
on the previous experiment.
Assessments
in the Rules for Seed Analysis for the studied species.
the 10th day for the end of the test. The percentage of
germination (GERM) and abnormal seedlings (AS),
(GSI), mean germination time (MGT), seedling length
(SL) and seedling dry mass (SDM) were considered. For
SL and SDM, the seedling as a whole was considered,
that is, with aerial part and root.
The seeds were placed to germinate on blotting paper
moistened with distilled water in the amount of 2.5 times
the mass of the non-hydrated paper and placed in crystal
polystyrene boxes (Gerbox® (DicaLab – Londrina-PR))
with dimensions 11 cm × 11 cm × 3 cm. The Gerbox®
25°C for a photoperiod of 16 hr and under an illuminance
of 120 µmol s–1 m–2
2009).
The GERM was determined considering the normal
seedlings, and the AS were also evaluated (BRASIL,
2009), being considered normal those that showed the
potential to continue their development and give rise
to normal plants when developed under favourable
conditions. The FGC was performed at the time of
primary root protrusion, which occurred on the 4th
percentage.
Concomitantly to the germination test, the count of
germinated seeds was performed to establish the GSI
obtained through the formula described by Maguire
(1962). GSI = G1/N1 + G2/N2 + … + Gn/Nn, in which G1,
G2 and Gn are the number of normal seedlings, computed
N1, N2 and Nn are the number of sowing days at the
was obtained through the methodology described by
Labouriau (1983) and performed simultaneously with
the germination test, the number of germinated seeds
being counted daily. This index is calculated by the MT
equation = (G1T1 + G1T1 + … + GnTn)/(G1 + G2 + … + Gn),
where MT is the mean time, in days, required to reach
4 Free and nanoencapsulated gibberellic acid in the germination of Dyckia sp.
maximum germination; G1, G2 and Gn are the number
of germinated seeds at times T1, T2 and Tn, respectively.
At the end of 10 days, the GERM and AS were
determined, and the result was expressed as percentage,
in addition to the SL (mm) through the measurement of
normal seedlings and obtained with the aid of a calliper.
The SDM (mg) was determined on an analytical scale
paper bags and ovens with forced air circulation at 65°C
until they reached constant mass.
Imbibition curve
Aiming to identify the phases of the germination
process, the seeds were weighed at intervals of 1 hr (in
from the beginning of the weighing. The seeds were
placed on blotting paper moistened with distilled water,
in a volume 2.5 times the dry weight of the paper and
placed in Gerbox® boxes. In the treatment with GA3,
L–1 for the species D. cabrerae and D. pottiorum and
5.0 mg L–1 for D. dusenii and D. walteriana. Seeds
without treatment with GA3
water for the same 5 min. Next, the seeds were placed
on blotting paper. Before obtaining the mass of the seeds
paper, and the mass was obtained on a semi-analytical
scale.
Experimental design and statistical analysis
The experimental design was completely randomised in
5.0 mgL–1
treatments (free GA3, NP ALG/CS/GA3, NP ALG/CS,
NP CS/TPP/GA3 and NP CS/TPP) with seven doses in
each one (0, 0.25, 0.5, 0.75, 1.0, 2.5 and 5.0 mg L–1).
For the germination test of the two stages and imbibition
curve, at each dose, four repetitions of 50 seeds were
used. For measurements of length and dry mass, 16
seedlings were randomly selected per replication, and
the result was expressed as the average of the four
seedlings.
The assumptions of normality of errors and
homogeneity of variances were tested using Shapiro–
p
the data were subjected to analysis of variance at
observed, linear or non-linear regression analysis
was performed (quadratic, 3- or 4-parameter logistic,
segmented, Brain–Cousens logistic model). To obtain
the imbibition curves, the data were adjusted using cubic
(Shimizu et al., 2022) of R software was used (R Core
Team, 2022).
RESULTS
Lot characterisation
For the characterisation of the seed lot, the values
of viability and water content of the seeds are shown
in Table 1. The four species studied presented high
viability immediately after harvesting, ranging from
78% to 88%, and germination from 71% to 84%. After
storage for 270 days in a dry and cold place, there
was a reduction in viability to values between 68%
and 73% and germination to 36% to 45%. In addition,
the water content remained low after storage, ranging
from 8.4% to 9.5% after harvest to 7.7% to 8.9% after
storage. However, it is noteworthy that a reduction in
time were observed, indicating a possible induction of
dormancy caused by the storage period. In addition,
the conservation period and in the germination test.
Eect of GA3 on Dyckia species
Table 2 presents the p-value of the analysis of variance of
the evaluated variables of bromeliad seeds (D. cabrerae,
D. dusenii, D. pottiorum and D. walteriana) according
3. There was a response to
GA3 doses for all species. AS did not respond to GA3
in any of the species, ranging from 2% to 4%. For
D. cabrerae
doses of GA3. For D. pottiorum, there was no response
for GERM, MGT, SL and SDM, while D. walteriana
and D. dusenii responded to all variables (except AS).
D. cabrerae showed an increase in SDM up to the
estimated dose of 1.2 mgL–1, obtaining a mass of 4.5
mg at this dose, and from this point on, there was a
reduction. In the control, for example, the SDM was 3.1
mg (Figure 1). For D. dusenii, all variables, except AS,
showed responses under GA3 doses (Figure 2).
Table 1. Viability by tetrazolium test (%), germination (%) and water content (%) of Dyckia spp. after harvest (10 days)
and after storage (270 days).
Species Viability Germination Water content
10 days 270 days 10 days 270 days 10 days 270 days
Dyckia cabrerae 79 68 71 38 9.2 8.5
Dyckia dusenii 81 71 75 36 9.5 8.9
Dyckia pottiorum 88 69 84 45 8.7 8.1
Dyckia walteriana 78 73 75 37 8.4 7.7
Baudraz de Paula et al. 5
Table 2. p-value of the F test of the analysis of variance and CV (%) for the variables: FGC (%), GERM (%), AS (%),
GSI, MGT (days), SL (mm) and SDM (mg) of seedlings for the species Dyckia cabrerae, D. dusenii, D. pottiorum and
D. walteriana as a function of doses of GA3.
D. cabrerae
FGC GERM AS GSI MGT SL SDM
p-value 0.8012 0.8558 0.9081t0.8954 0.7463 0.8915 <0.0 01
CV (%) 41.14 16. 33 50.42 7.35 4.86 5.49 10.10
D. dusenii
p-value 0.0037 <0.001 0.9844t<0.001 0.0211 0.0011 <0.001
CV (%) 26.34 11.45 40.2 13.00 5.40 8.65 11.08
D. pottiorum
p-value 0.0102 0.3135 0.8692t0.9646 0.0166 0.0403 <0.001
CV (%) 23.44 14.95 37.86 17.23 7.81 16.02 14.80
D. walteriana
p-value <0.0 01 <0.001 -<0.001 < 0.001 <0.0 01 <0.001
CV (%) 15.80 6.00 -5.18 3.16 2.89 5.78
t
3, gibberellic acid; GERM, germination;
GSI, germination speed index; MGI, mean germination time; SDM, seedling dry mass; SL, seedling length.
Figure 1. Dyckia cabrerae SDM as a function of GA3
doses. GA3, gibberellic acid; SDM, seedling dry mass.
For FGC, GERM and SDM, the highest values found
were at a dose of 5.0 mg L–1 (21%, 62% and 3.1 g,
respectively). In comparison, in the control treatment,
the values for the same variables were 10%, 36% and
1.8 g, respectively. MGT was lower at a dose of 5.0 mg
L–1
germination.
There was an increase in the GSI with the increased
dose of GA3; however, this index stabilised at higher
doses, ranging from 9.53 to 11.08 between doses of
1.0 mg L–1 and 5.0 mg L–1. A similar response was
obtained for SL, so that at doses between 1.0 mg L–1
and 5.0 mgL–1, seedlings of 7.65 mm and 7.97 mm were
obtained. The responses of D. pottiorum to GA3 doses
for the variables MGT, FGC, SL and SDM are described
in Figure 3.
For FGC, there was an increase in germination up
to a dose of 0.8 mgL–1 with 15% germinated seeds.
Above this dose, there was a reduction in germination
and stabilisation from a dose of 1.0 mg L–1, ranging
from 9% to 11%. For MGT, doses of 0.75 mgL–1 and 1.0
mgL–1 provided a shorter time for seed germination,
with 3.20 days and 3.38 days, respectively.
In SL, there was an increase of up to a dose of 1.2
mgL–1, reaching 7.49 mm, with subsequent reduction
in length. For SDM, the estimated dose of 0.7 mgL–1
showed greater mass, with 5.7 mg, and showed a mass
reduction for doses above this value. For these variables,
in relation to the maximum point obtained, there was an
increase of 46% in SL and 56% in SDM compared to
the control. D. walteriana responded to all variables at
3 (except AS) (Figure 4).
For FGC and GERM, the maximum point obtained
was at a dose of 5.0 mg L–1, with 23% and 64%,
respectively. By comparison, the control presented 10%
FGC and 35% GERM. In the GSI variable, the highest
value was also observed for a dose of 5.0 mgL–1 (with
10.88), as well as the lowest value of MGT (2.98). The
same was also observed for the growth variables, with a
greater response in SL (8.28 mm) and SDM (3.98 mg) at
a dose of 5.0 mgL–1.
Regarding the imbibition curves, all the species
studied showed three-phase water absorption behaviour,
with a rapid increase in initial mass, subsequent
stabilisation and then a new increase. According to
Figure 5, for D. cabrerae, phase I lasted 50 hr, and phase
III, that is, germination itself, started at 92 hr. On the
6 Free and nanoencapsulated gibberellic acid in the germination of Dyckia sp.
Figure 2. GERM (A), GSI (B), MGT (C), FGC (D), length (SL) (E) and SDM (F) of Dyckia dusenii as a function of
GA33, gibberellic acid; GERM, germination; GSI, germination speed index;
MGT, mean germination time; SDM, seedling dry mass; SL, seedling length.
other hand, when treated with GA3, phase I lasted 46 hr,
and germination started after 90 hr. Phase II lasted 42
hr without treatment with the regulator and 44 hr with
the use of the GA3.
For D. pottiorum, phase I lasted 46 hr, and phase III
started at 88 hr, while for seeds treated with GA3, phase
I was shorter, lasting 34 hr, and germination started at
90 hr. Phase II lasted 56 hr in treated seeds and 42 hr
in seeds not treated with GA3. For both species, it was
possible to observe that even with the GA3 treatment,
there was no great variation in the beginning of
germination.
The imbibition curves for D. dusenii and D.
walteriana had similar behaviour to the cited species.
However, they were responsive to pre-treatment
with GA3, which reduced the time for the onset of
germination, as shown in Figure 6.
For D. dusenii, phase I lasted 42 hr, while with GA3,
the time was reduced to 34 hr. Phase II lasted 50 hr for
treated seeds, while for untreated seeds, it lasted 48 hr.
Germination (phase III) started after 90 hr without GA3,
while with GA3, it started after 84 hr.
D. walteriana species had phase I lasting 42 hr,
which was reduced to 34 hr in seeds treated with GA3.
Phase III, on the other hand, started with 86 hr in the
treatment with water only, reducing to 82 hr in the
treated seeds.
Eect of nanoencapsulated GA3 on D. walteriana
3 on
germination and vegetative growth, mainly in relation
to D. dusenii and D. walteriana, only the latter was
chosen for the nanoencapsulated GA3 tests. The option
for D. walteriana3
Baudraz de Paula et al. 7
Figure 3. MGT (A), FGC (B), length (SL) (C) and SDM (D) of Dyckia pottiorum as a function of GA3
germination count; GA3, gibberellic acid; MGT, mean germination time; SDM, seedling dry mass; SL, seedling length.
on this species, due to its occurrence in the region where
the study was carried out, and due to the higher degree
of threat of extinction in which the species is found.
Through analysis of variance, it was possible to
according to the treatment. The only exception was BP,
which had a variation of 2–3% in all treatments. It was
also possible to observe that treatments with only NPs
FGC, MGT, SL and SDM (Table 3).
For FGC, the maximum point obtained for free
GA3 was at a dose of 5 mgL–1 with 23%, while in the
treatments with nanoencapsulated GA3, it was 19% and
18%, in the estimated doses of 1.4 mgL–1 and 0.3 mg
L–1 for NP ALG/CS-GA3 and CS/TPP-GA3, respectively.
In GERM, the highest response was obtained for free GA3
with 64% at a dose of 5 mgL–1, and when encapsulated,
the maximum response was 60% for NP ALG/CS-GA3
at an estimated dose of 2.1 mg L–1 and 55% for NP
CS/TPP-GA3 at an estimated dose of 1.7 mgL–1. The
control treatment presented 35% GERM and 10% FGC
(Figure 7).
In the GSI and MGT variables, the highest values
were also observed for free GA3 at a dose of 5 mgL–1,
with 10.88 and 2.98, respectively. For treatments with
nanoencapsulated GA3, the estimated dose of 0.5 mgL–1
presented the h ighest values of GSI and MGT, respectively,
of 10.19 and 3.16 for NP ALG/CS-GA3 and of 9.05 and
3.28 for NP CS/TPP-GA3. The control treatment presented
GSI and MGT, respectively, of 7.05 days and 4.16 days.
For MGT, it was also possible to observe a response from
empty NPs (NP ALG/CS and CS/TPP), with a maximum
response at a dose equivalent to 2.9 mg L–1 for NP ALG/
CS with 3.8 days, and 2.7 mgL–1 for NP CS/TPP with 3.8
days (Figure 8).
For SL, when seeds were treated with free GA3, the
dose of 5.0 mgL–1 showed the best response, obtaining
average seedlings of 8.28 mm. The same was observed
for SDM, with 3.98 mg for the same dose. For NP ALG/
CS-GA3, the estimated optimum dose was 0.3 mgL–1
with 8.04 mm of SL and 3.68 mg of SDM. For NP CS/
TPP-GA3, the estimated dose of 0.2 mg L–1 had the best
response for SL with 7.78 mm, and the estimated dose
of 0.4 mgL–1 presented higher SDM with 3.45 mg. It
was also possible to observe an empty response for NP
CS/TPP for SL, with 7.26 mm at the equivalent dose of
2.0 mgL–1. The control treatment presented a mean SL
of 6.61 mm and an SDM of 2.15 mg (Figure 9).
DISCUSSION
Lot characterisation
Dyckia seeds are considered orthodox, meaning that
they are able to tolerate low humidity levels and storage
8 Free and nanoencapsulated gibberellic acid in the germination of Dyckia sp.
Figure 4. GERM (A), GSI (B), MGT (C), FGC (D), length (SL) (E) and SDM (F) of Dyckia walteriana as a function
of GA3 3, gibberellic acid; GERM, germination; GSI, germination speed index;
MGT, mean germination time; SDM, seedling dry mass; SL, seedling length.
at cool temperatures. Thus, it is essential to maintain
adequate conditions to ensure seed viability for a long
period (Zucchi et al., 2018). In addition, the maximum
such as the origin of the seeds and environmental
conditions during the maturation, harvesting and drying
process. Therefore, it is important to understand the
physiological behaviour and the ideal storage conditions
for the conservation of seed viability (Zucchi et al.,
2018; Fior et al., 2020).
In addition to the storage conditions for seeds of the
species under study being recommended for seeds with
orthodox behaviour (cold and dry), the ideal period for
storing and maintaining seed viability is directly related
to the water content in which the seeds are conserved. In
this sense, t he water content, despite being at an adequate
level for the studied species (7%–9%), is not applicable
a tolerable limit regarding desiccation (Rajanaidu and
Ai nul, 2013).
Even under ideal storage conditions, there was
a loss of viability of these seeds since with aging,
damage to the membranes occurs and the enzymes
lose their catalytic activity (Oliveira et al., 2011). In
addition, according to the observed results, the seeds
may have entered a state of dormancy after storage.
This can also be explained by the tetrazolium test,
where despite the low germination after storage, the
seeds presented high viability (71–84%). A favourable
point of the tetrazolium test is that its results do
interfere in germination analysis, such as the incidence
of microorganisms; in addition, this test also serves to
clarify factors not explained by the germination test,
Baudraz de Paula et al. 9
Figure 5. Curve of water absorption by the seeds of Dyckia cabrerae without (A) and with GA3 (B) and D. pottiorum
without (C) and with GA3 (D) at a dose of 0.75 mg L–1. GA3, gibberellic acid.
in this case, relating to dormancy (Carvalho et al.,
2013).
Another associated factor is that most Dyckia species
present high germination at higher temperatures,
thus, the induction of dormancy under these storage
conditions (cold) could have occurred. Dormancy can
be characterised as a failure of germination even under
apparently favourable conditions for germination. In
this case, the dormancy can be divided into primary or
secondary. Primary dormancy occurs during the seed
maturation phase, that is, it is dispersed already in a
dormant state (Silva et al., 2018).
In the case of the seeds under study, this primary
as secondary dormancy. All the processes involved in
this type of dormancy are not yet well elucidated in the
literature, and it is recognised that seeds with secondary
dormancy germinate normally, but when exposed to
unfavourable environmental factors, they are induced
to a state of dormancy (Silva et al., 2018). Among the
possibilities of dormancy, the physiological possibility
occurs through the interaction between inhibitors and
germination promoters and is generally overcome
through the addition of plant growth regulators, such
as GA3, which was observed in this study (Rego et al.,
2018).
Eect of GA3 on Dyckia species
D. dusenii and D. walteriana showed stimulation in the
germination process. In this sense, gibberellins (GAs)
have the ability to promote seed germination. Thus, a
high level of GAs and low level of ABA is a favourable
condition for seed germination (Tuan et al., 2018;
Zhong et al., 2021). As germination progresses, seed
reserves are gradually degraded, providing energy and
metabolites for germination and seedling establishment
(Xiong et al., 2021).
This GA-induced degradation of seed reserves
occurs through the production of hydrolases, which
the embryo (Bocatto and Forti, 2019). Hydrolases,
such as amylases, react with the reserves stored
substances, allowing the resumption of growth of
the embryonic axis. With this, simple sugars, amino
acids and nucleic acids are formed that stimulate cell
accelerating and standardising germination (Paixão
et al., 2021).
10 Free and nanoencapsulated gibberellic acid in the germination of Dyckia sp.
Figure 6. Water absorption curve by Dyckia dusenii seeds without (A) and with GA3 (B) and D. walteriana without
(C) and with GA3 (D) at a dose of 5.0 mg L–1. GA3, gibberellic acid.
Table 3. p-value analysis of variance and CV (%) for the variables: FGC (%), GERM (%), AS (%), GSI, MGT (days),
SL (mm) and SDM (mg) of seedlings for treatments NP ALG/CS-GA3, NP CS/TPP- GA3, NP ALG/CS and NP CS/
FGC GERM AS GSI MGT SL SDM
NP ALG/CS-GA3
p-value <0.0 01 <0.001 -<0.001 <0.0 01 <0.001 <0.0 01
CV (%) 14. 56 5.41 -3.41 3.65 2.35 7.09
Mean - - 3.14 - - - -
NP CS/TPP-GA3
p-value <0.0 01 <0.001 -<0.001 <0.0 01 <0.001 <0.0 01
CV (%) 14.97 7.24 -4.23 3.27 1.97 6.44
Mean - - 2.29 - - - -
NP ALG/CS
p-value 0.414 0.064 -<0.001 0.024 0.053 0.474
CV (%) 22.68 10.74 -6.08 3.20 4.70 11.18
Mean 11.0 42.0 2 .71 7.45 -7.0 6 7.09
NP CS/TPP
p-value 0.669 0.057 -<0.001 0.006 0.003 0.406
CV (%) 22.09 11.18 -3.49 2.56 2.72 8.28
Mean 11.0 38.0 3.14 7.06 - - 2.24
count; GA3, gibberellic acid; GERM, germination; GSI, germination speed index; MGT, mean germination time; NP, nanoparticle; SDM,
seedling dry mass; SL, seedling length; TPP, sodium tripolyphosphate.
Baudraz de Paula et al. 11
Figure 7. GERM (A) and FGC (B) of Dyckia walteriana
acid formulation (GA3 3 gibberellic acid;
GERM, Germination; NP, nanoparticle; TPP, sodium tripolyphosphate.
Figure 8. GSI (A) and MGT (B) of Dyckia walteriana
acid formulation (GA3). ALG, sodium alginate; CS, chitosan; GA3 gibberellic acid; GSI, germination speed index;
MGT, mean germination time; NP, nanoparticle; TPP, sodium tripolyphosphate.
Figure 9. Length (SL) (A) and dry mass (SDM) (B) of Dyckia walteriana
and types of gibberellic acid formulation (GA3). ALG, sodium alginate; CS, chitosan; GA3 gibberellic acid; NP,
nanoparticle; SDM, seedling dry mass; SL, seedling length; TPP, sodium tripolyphosphate.
12 Free and nanoencapsulated gibberellic acid in the germination of Dyckia sp.
It was observed in D. cabrerae and D. pottiorum
that GA3 did not improve germination parameters, as
also observed by Pompeli (2006) in studies with seeds
of Dyckia encholirioides (Gaudichaud) Mez., verifying
that the application of GA3 did not result in the germinal
stimulus. The fact that these species respond in growth,
but not in germination, is an indication that GA3 is not
acting in the embryo, either because it cannot get in
contact with it or because it is inact ivated. However, after
radicle protrusion, there is contact with the regulator,
development, in this case, altering the concentrations of
gibberellins (GAs) and/or altering the plant’s ability to
respond to this plant growth regulator (Kashiwaqui et
al., 2019).
The regulation of endogenous levels of GAs in
seeds, as well as in the plant, occurs through several
processes. Internally, the so-called active GAs undergo
the process of conjugation, t hat is, sugars such as glucose
thus reversibly inactive. GAs can also be irreversibly
inactivated by enzymes. These mechanisms control
and contribute to the balance in the levels of GAs; in
addition, studies demonstrate several inhibitors that
act in the process of biosynthesis of GAs (Kashiwaqui
et al., 2019).
Regarding the improvement in plant growth
variables, that is, length (SL) and mass (SDM), all
species were stimulated by GA3. Gibberellins promote
these responses through several factors, such as the
orientation of microtubules towards the axis of cell
growth; thus, cells increase only towards the axis of
growth (Chaudhary et al., 2019; Purba et al., 2021).
GAs stimulate cell division, especially meristematic
Furthermore, GAs have the potential to stimulate active
plant growth, such as stem and leaf elongation. GAs also
2013; Chaudhary et al., 2019; Purba et al., 2021).
Research shows that the use of GA3
100 µM of GA3 promoted greater height and a greater
Impatiens hawkeri
of lisianthus leaves (Eustoma grandiorum (R a f.)
Shinners) with the application of 150 ppm of GA3.
The pre-germination treatment of beton seeds
(Rhaphiodon echinus Shauer.) with GA3 at concentrations
of 0.5 and 1.0% increased the germination process
and decreased the average germination time (Souza et
al., 2018). The treatment of imperial bromeliad seeds
Alcantarea imperialis (Carriere) Harms (Bromeliaceae)
with 5.0 mgL–1 GA3 promoted greater germinability in
relation to the other treatments (Bonin et al., 2010).
The imbibition curve data indicate a triphasic
that the speed of occurrence of this process depends
on the characteristics of the seeds of each species, such
as the chemical composition and permeability of the
seed coat (Albuquerque et al., 2009). Phase I results in
rapid absorption of water and is characterised by the
the seeds to guarantee energy and essential nutrients for
the growth of the embryo (Araújo et al., 2018).
In phase II, water absorption decreases and
stationary. Another characteristic of this phase is the
reactivation and i nitiation of cellular meta bolic processes,
with the expansion of the embryo and increased
activity of enzymes used for embryo development.
Finally, in phase III, there is again an increase in water
absorption by the seed due to seedling growth and cell
is that germination becomes visible, that is, the radicle
protrudes (Araújo et al., 2018).
Morphological studies carried out by Duarte et
al. (2009) in D. goehrinii Gross & Rauh suggest the
the seed coat, allowing the nucella to become highly
vascularised. This characteristic observed in this species
in Dyckia species; however, studies are needed for this
According to the i mbibition cur ve, none of the species
of Dyckia (D. cabrerae, D. dusenii, D. pottiorum and
D. walteriana) presented seeds with dormancy, at least
the integument. Tegumentary dormancy, considered a
mechanical restriction, is discarded due to the fact that
seeds, that is, the seeds were able to absorb water. Thus,
the possibility is that the seeds had entered secondary or
physiological dormancy (Rego et al., 2018), which is also
evidenced by the positive response generated by GA3.
It is also noted that GA3 acted on the embryo of the
most responsive species (D. dusenii and D. walteriana)
by accelerating the germination process through
reducing the imbibition phases. On the other hand, in
D. cabrerae and D. pottiorum seeds, GA3 only reduced
and percentage of germination. These species were
responsive to GA3 in parameters related to the seedlings,
suggesting that some internal mechanism of the embryo
inactivated GA3 and, after germination, the radicles
of the seedlings, when in direct contact with the GA3,
and mass, thus responding to the regulator (Kashiwaqui
et al., 2019).
Eect of nanoencapsulated GA3 on D. walteriana
The purpose of application through NPs is to provide
slow and sustained release of the active substance,
in addition to protecting it against degradation
Baudraz de Paula et al. 13
(Pereira et al., 2017). One of the advantages of using
biodegradable polymeric systems is that they can
be used in the metabolism of living organisms.
Furthermore, in this study, none of the NP systems
phases, corroborating the results found in the literature
(Pereira et al., 2019).
It was observed that the responses for freely applied
and nanoencapsulated GA3
response points, demonstrating that the form of
at lower doses (0.75mg L–1 and 1.0 mg L–1), while
3 in the free form occurred at
the highest doses (2.5 mgL–1 and 5.0 mgL–1). This
can be explained by the fact that free GA3 had direct
contact with the seeds, being released faster than
nanoencapsulated GA3, which provided a slower release.
In addition, some NPs remain adhered to the surface of
the seed, maintaining the supply of GA3 even after the
adsorbed by the cell wall (Pereira et al., 2019).
The CS present in NPs is easily absorbed, prolonging
the contact time and facilitating the absorption of
bioactive molecules, in this case GA3. The good
acceptability and absorption of NPs are also reported
due to the association with the surface of the plant,
or seed, in this case, due to the presence of carboxyl,
hydroxyl, amide and phosphate groups that provide
potential sites for binding with CS, which with its
cationic properties, manages to adsorb on the surface,
prolonging the contact of the nanoparticle with the plant
(Li et al., 2019).
In the study in question, there was no better response
with the use of nanoencapsulated GA3, with emphasis
on the dose reduction when supplied this way, probably
due to better delivery of GA3 to the plants. The fact
can be explained by the fact that the study experiment
was carried out in a fully controlled environment
degradation and bioavailability of GA3.
containing GA3 through alginate/chitosan (ALG/CS)
and CS/TPP, it was demonstrated that the treatment
of seeds with ALG/CS-GA3
provided an increase in root development, leaf area and
photosynthetic pigments (chlorophylls and carotenoids)
of bean plants (P. v ulgaris L.) (Pereira et al., 2017).
The same resu lt was also found in the present stu dy, i n
which NPs containing ALG/CS showed a better response
than CS/TPP, which was also observed by Pereira et al.
(2019), in which tomato seeds (S. lycopersicum var.
better responses to the NPs of ALG/CS-GA3, which
provided an increase in the dry mass of shoots and roots
and photosynthetic pigments, while the CS/TPP-GA3
formulation showed relatively low biological activity
during the initial growth of the plants.
3 release
NP characteristics, the zeta potentials of the studied
positive for CS/TPP. Studies show that the zeta potential
participates in the plant–NP interaction. NPs with
positive zeta potential, such as CS/TPP, have a strong
interaction with the negative groups of the plant cell
wall, with low cell internalisation and a tendency to
accumulate on the cell surface. On the other hand, NPs
with negative zeta potential, such as those of ALG/CS,
manage to be rapidly distributed and internalised in
cells (Zhu et al., 2012).
CS/TPP was observed for some variables. Of the
compounds used in the formulation of NPs, the ability
of CS to increase the response in plants depending on
the species and concentration has already demonstrated
in several studies, being mainly associated with plant
(Malerba and Cerana, 2016; Odat et al., 2021).
Chitosan was able to improve seed germination and
initial growth of wheat seedlings (Triticum aestivum
hypocotyl and radicle length and dry mass in relation to
control vetch seeds Vicia sativa L. (Odat et al., 2021).
The present study shows the importance of
and during storage and that seeds of Dyckia species,
with the aid of plant growth regulators, return to good
germinability even after a long storage period and a
reduction in germination potential. In addition, the
studies regarding the acceptance of NPs by the seed and
the use of nanotechnology in several areas. These results
are promising in the sense of bringing information
and possibilities for the cultivation of this group of
predominantly native plants and inserting them in the
CONCLUSIONS
For D. dusenii, a dose of 5.0 mgL–1 is recommended,
and for D. walteriana, it is between 4.4 mg L–1 and
5.0 mg L–1 of GA3. The species D. cabrerae and D.
pottiorum responded only to vegetative growth, GA3 not
being recommended for germination.
When nanoencapsulated, GA3 resulted in greater
responses at lower doses, proving the controlled
release of the regulator, with greater responses for
NPs containing ALG/CS. The dose of 5.0 mg L–1 of
free GA3 and between 0.75 mg L–1 and 1.0 mg L–1
for nanoencapsulated containing ALG/CS-GA3 are
recommended for germination of D. walteriana.
14 Free and nanoencapsulated gibberellic acid in the germination of Dyckia sp.
FUNDING
for the Improvement of Higher Education Personnel
(n° 88882.448347/2019-01), and the National Council
301684/2017-0 and 311034/2020-9, respectively).
AUTHOR CONTRIBUTIONS
J.C.B.P and H.C.O. – conceptualisation. J.C.B.P., H.R.G.
and G.D.S. – methodology. H.C.O., J.C.B.P., R.T.F.,
L.F.F. and A.E.S.P. – validation. J.C.B.P., H.C.O., G.D.S.
and K.A.M.M. – investigation. G.D.S. – data curation.
J.C.B.P., H.C.O., G.D.S. and K.A.M.M. – writing –
original draft preparation. H.C.O., R.T.F., L.F.F. and
A.E.S.P. – writing – review and editing. H.C.O. –
supervision. All authors have read and agreed to the
published version of the manuscript.
CONFLICT OF INTEREST
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Received: August 14, 2023; accepted: October 23, 2023
