plants

Article
Light and Ethephon Overcoming Seed Dormancy in Friar’s
Crown (Melocactus zehntneri, Cactaceae), a Brazilian Cactus
Mariana Freitas Campos Magnani 1, * and Jean Carlos Cardoso 2

1 Research Group in Physiology Applied to Agriculture, Center of Agricultural Sciences, Federal University of
São Carlos, Rodovia Anhanguera, km 174, Araras 13600-970, SP, Brazil
2 Laboratory of Plant Physiology and Tissue Culture, Department of Biotechnology Plant and Animal
Production, Center of Agricultural Sciences, Federal University of São Carlos, Rodovia Anhanguera, km 174,
Araras 13600-970, SP, Brazil; [email protected]
* Correspondence: [email protected]

Abstract: Seed germination in Melocactus and other cactus species is hampered by dormancy. How-
ever, most studies failed to achieve high seed-germination rates, suggesting a complex mechanism of
dormancy in Cactaceae. Thus, the objective of this study was to evaluate whether factors such as
light and phytoregulators overcome the dormancy in the seeds of the friar’s crown cactus (Melocactus
zehntneri). Two consecutive experimental sets were designed: one with seed germination under
filter paper conditions and different wavelengths and Photosynthetically Photon Flux Densities
(PPFDs); and one in vitro experiment using a culture medium to evaluate the influence of different
phytoregulators, such as gibberellic acid (GA3 ), benzylaminopurine (BAP) and ethephon (ET), both in
the germination of seeds of M. zehntneri. Seeds of M. zehntneri are positive photoblastic. Red light and
gradual increases in PPFD resulted in the highest germination rates (60.8–61.7%) and germination
speed index (4.4–4.5). In vitro seeding in culture media increased the germination percentage to 76%
in control without phytoregulators. Ethephon showed a major effect in releasing the germination
of dormant seeds of M. zehntneri, totaling 98% of seeds germinated under in vitro conditions, while
GA3 and BAP showed minor or no effect on germination. The present study resulted in an efficient
in vitro technique for germination and a better understanding of cacti seed dormancy.

Keywords: cactus; dormancy breaking; germination; light; phytoregulators

Citation: Magnani, M.F.C.; Cardoso,
J.C. Light and Ethephon Overcoming
Seed Dormancy in Friar’s Crown
(Melocactus zehntneri, Cactaceae), a
Brazilian Cactus. Plants 2023, 12, 1. Introduction
4127. https://doi.org/10.3390/ The genus Melocactus comprises plants popularly known as ‘chapéu-de-frade’ or friar’s
plants12244127 crown cacti, and Brazil is the country with the highest diversity, with 55% of the number of
Academic Editor: Alberto Gianinetti
Melocactus species in the world occurring throughout the northeast region, and in parts of
the north and southeast regions of Brazil [1].
Received: 11 October 2023 The demand for Melocactus species in the commercial sector, especially for ornamen-
Revised: 6 December 2023 tation and collecting purposes, has had remarkable growth in recent years, due to its
Accepted: 7 December 2023 peculiar aesthetics, ornamentation, and easy cultivation. However, it is difficult to be pre-
Published: 11 December 2023
cise about the exact numbers of the trade of these plants since the illegal trade is difficult to
quantify [2]. The fact is that many Melocactus populations have been drastically reduced [3].
Consequently, some Melocactus species are already under protection as a way of controlling
Copyright: © 2023 by the authors. the high extraction for trade [4,5]. The species M. zehntneri is classified as a least concern
Licensee MDPI, Basel, Switzerland. species, but its population has also shown a recent and constant decline and has recently
This article is an open access article been placed under protection [6,7]. In part, its population reduction can be attributed to two
distributed under the terms and main factors: the continuous deforestation of the Caatinga Biome and the replacement of
conditions of the Creative Commons wild species with agriculture and livestock activities, and the recent increase of commercial
Attribution (CC BY) license (https:// exploitation resulting in the extraction of individuals for the production of food, traditional
creativecommons.org/licenses/by/ medicines, and animal fodder [8,9], but especially for ornamental purposes [10].
4.0/).

Plants 2023, 12, 4127. https://doi.org/10.3390/plants12244127 https://www.mdpi.com/journal/plants

Plants 2023, 12, 4127 2 of 13

Melocactus displays a distinctive feature unique to the genus known as a cephalium at

its apex. This characteristic occurs only when the cactus reaches reproductive maturity at
approximately 10 years of age, mainly due to very slow vegetative growth (0.04–4.7 cm
per year) [11]. The natural reproduction is exclusively by seeds due to limited vegetative
propagation since they do not develop segmentations as observed in other cactus species [6].
In addition, seed germination in different species of Melocactus is hampered by dormancy.
Different studies have reported the use of chemical scarification to increase germination
rates and associated dormancy with the rigid seed coat. Also, phytoregulators, such as
gibberellic acid (GA3 ), have been used to increase the germination rate in Melocactus. In
M. azureus, the imbibition of seeds in water or gibberellic acid (GA3 ) for 2 h resulted in
an increase of 14 and 20% seeds germinated, compared to only 3% germinated seeds in
the control [12]. However, studies with other phytoregulators aiming to overcome seed
dormancy are very limited. On the other hand, the key role of ethylene in the dormancy
release of seeds of numerous plant species has been reported [13].
The use of in vitro germination has been proposed as an alternative to increase germi-
nation rates in Melocactus. Successful in vitro germination was reported for M. glaucescens
(68.1%), M. sergipensis (64%), M. zehntneri (58.7%), and M. violaceus (59.3%) [14]. Addition-
ally, previous results obtained by our group have demonstrated similar percentages of
seeds germinated in Petri dishes (50–55% on average) for M. zehntneri [15]. Thus, the results
obtained with different species of Melocactus showed that a substantial part of the seeds
(40–80%) continues with some unknown type of dormancy, with no germination.
Thus, the present study aimed to test and determine the main factors affecting the
germination of M. zehntneri seeds: the wavelength, light intensity, and phytoregulators,
such as Gibberellic acid (GA3 ), Benzylmaninopurine (BAP), and Ethephon. From a practical
point of view, the experiments aim to develop an efficient protocol for seed germination.
This protocol serves two purposes: to enable in vitro conservation and to serve as a method
for propagation of the Melocactus species, which could offer insight for conducting further
studies on breaking seed dormancy in Cactaceae.

2. Results
2.1. Effects of Light Intensity and Wavelength on Germination
The percentage of seeds germinated using filter paper was affected by wavelength
and PPFD, but the interaction between these two factors was non-significant. Thus, the
effects of light wavelength (Figure 1A,B) and PPFD (Figure 1B,C) were analyzed separately.
The highest germination percentage ranged from 60.8% to 61.7% and was reported in the
white, red, and red/blue LEDs. The use of only blue LEDs resulted in a drastic reduction
in seed germination to 37.5% (Figure 1A) and the lowest GSI values (Figure 1B). About
the PPFD, the gradual increases of PPFDs of light resulted in a positive and significant
correlation with the percentage of germinated seeds of M. zehntneri (Figure 1C). The GSIs
values were also increased in the higher PPFDs (II and III), compared to the lowest PPFD
used (Figure 1B).
The germination of the first seeds was reported 4 days after seeding (DAS) in the
red/blue and white LEDs and at 7 DAS in the other wavelengths. The blue light resulted
in the late beginning of germination at 9 DAS, with the lowest average germination speed
(AGS) (Figure 1D). For the red, white, and red/blue LEDs, the maximum AGSs occurred
between DAS 9 and 14 (Figure 1D). Increases in PPFD were those resulting in the highest
values of AGS. The different wavelengths also affected the color of embryos after ger-
mination. In the red light, 100% germinated embryos had a light green color, related to
chlorophyll biosynthesis; in the blue and white LEDs, the red–purple color was predom-
inant in the embryos. Intermediately, the presence of green and purple embryos in the
red/blue LED was also observed (Figure 2).
23, 12, x FOR PEER REVIEW
Plants 2023, 12, 4127 3 of 13

Figure 1. Germination
Figure 1. Germination (%)(%) of seeds
of seeds of Melocactus
of Melocactus zehntneri zehntneri cultivated
cultivated under under
different light different ligh
wavelengths
lengths and
andPPFDs
PPFDs sourced
sourced by LEDs:
by LEDs: percentage
percentage of seed (A)
of seed germination germination (A) (Different
(Different lowercase letters in lower
the same column indicate the effects of the wavelength and
ters in the same column indicate the effects of the wavelength and uppercase uppercase letters indicate the effects of
letters indi
PPFDs on germination. Shapiro–Wilk Normality Test = 0.36, Levene test for homoscedasticity = 0.71.
effects of PPFDs on germination. Shapiro–Wilk Normality Test = 0.36, Levene test for homo
Coefficient of variation = 13.2%. F-values for: wavelengths (10.81 **), PPFDs (4.86 *), and Interaction
ticity = 0.71. Coefficient
(1.67 ns). Significant of variation
at 5% (*) and 1%=(**)
13.2%. F-values
probability. for: wavelengths
ns, non-significant); (10.81
germination **),index
speed PPFDs (4.8
Interaction
(GSI)(1.67 ns). Significant
(B) (Shapiro-Wilk Normality at Test
5% =(*) and
0.50, 1% (**)
Levene probability.
test for ns, non-significant);
homoscedasticity = 0.68. Coefficient germ
speed index (GSI)= (B)
of variation (Shapiro-Wilk
24.95%. Normality
F-values for: wavelengths Test
(12.87 **),=PPFDs
0.50, (4.42
Levene
*), andtest for homoscedasticit
interaction (2.05 ns).
Significant at 5% (*) and 1% (**) probability. ns, non-significant); correlation
Coefficient of variation = 24.95%. F-values for: wavelengths (12.87 **), PPFDs (4.42 *), and int between the percentage
of seed germination and PPFD of light (C) and average germination speed under different LED
(2.05 ns). Significant at 5% (*) and 1% (**) probability. ns, non-significant); correlation betw
wavelengths (D).
percentage of seed germination and PPFD of light (C) and average germination speed unde
ent LED wavelengths (D). for the germination of M. zehntneri seeds drastically reduced the
The use of darkness
germination rate from 63.75% (control using light) to 11.3, 2.5, and 3.8% when seeds were
cultivated in darkness for 10, 20, and 30 days, respectively (Table 1). Interestingly, seeds
The germination of the first seeds was reported 4 days after seeding (DAS)
subjected to dark conditions for germination under short-period treatments, followed by
red/blue and white
exposure to lightLEDs andwere
conditions, at 7not
DAS ingerminate
able to the other forwavelengths.
up to 12 months. The blue light r
in the late beginning of germination at 9 DAS, with the lowest average germination
(AGS) (Figure 1D). For the red, white, and red/blue LEDs, the maximum AGSs oc
between DAS 9 and 14 (Figure 1D). Increases in PPFD were those resulting in the
values of AGS. The different wavelengths also affected the color of embryos after
nation. In the red light, 100% germinated embryos had a light green color, related t
rophyll biosynthesis; in the blue and white LEDs, the red–purple color was predo
in the embryos. Intermediately, the presence of green and purple embryos in the re
LED was also observed (Figure 2).
Plants 2023, 12, x FOR PEER REVIEW 4 of 14
Plants 2023, 12, 4127 4 of 13

Figure 2. Seed germination and color of germinated embryos of Melocactus zehntneri cultivated under
Figure 2. Seed germination and color of germinated embryos of Melocactus zehntneri cultivated un-
different
der light
different wavelengths:
light redred
wavelengths: and blue
and (R (R
blue + B); white
+ B); (W);
white redred
(W); (R)(R)
and blue
and (B).(B).
blue
Table 1. Percentages of seed germination in Melocactus zehntneri cultivated under different dark times
andThe use ofafter
measured darkness
45 daysfor
of the
seedgermination
maintenance of M. zehntneri
under seeds drastically reduced the
light conditions.
germination rate from 63.75% (control using light) to 11.3, 2.5, and 3.8% when seeds were
cultivated in darkness
Darkfor 10, 20, and 30 days, respectively
Time (Table of
Percentage 1).Germination
Interestingly, seeds
subjected Control
to darkunder
conditions for germination under short-period 63.75
light conditions treatments,
a followed by
exposure to light conditions, were not able to germinate for up to 12 months.
10 days 11.25 b
20 days 2.5 b
Table 1. Percentages of
30 seed
days germination in Melocactus zehntneri cultivated
3.8 bunder different dark
times and measured after 45 days of seed maintenance under light conditions.
F 31.386 *
Dark
CV(%) Time Percentage of Germination
84.223
Control under light conditions 63.75 a
* Means followed by different letters were significantly different by Tukey’s test at 5% probability. All germination
data were evaluated after10 exposing
days seeds to light conditions for 45 days (control, 45
11.25 b10 days dark + 45 days
days;
under light; 20 days dark + 45 days under light, and 30 days dark + 45 days under light).
20 days 2.5 b
2.2. Germination of M.30zehntneri
days Seeds In Vitro under Different Concentrations 3.8 b of Plant
Growth Regulators F 31.386 *
The germinationCV(%) percentage of M. zehntneri seeds using in vitro 84.223
conditions in a culture
* Means
medium followed
was 74%by different
(Figureletters were significantly
3), higher than observed different
usingby the
Tukey’s
same test at 5%
light probability.in
conditions
All
thegermination data were evaluated
previous experiment with Petri after exposing
dishes andseeds
filter to light (61.3%).
paper conditions for 45
Seed days (control,
imbibition in an
45ethephon
days; 10 days dark + 45 days under light; 20 days dark + 45 days under light,
solution as a pre-treatment resulted in a significant and striking increase in the and 30 days dark +
45 days under light).
percentage of in vitro germinated seeds of M. zehntneri. The best treatment and response of
seeds to ethephon was reported in the culture medium without phytoregulators, which
2.2. Germination of M. zehntneri Seeds In Vitro under Different Concentrations of Plant
resulted in 98% seeds germinated (Figure 3). In the control culture medium without the pre-
Growth Regulators
treatment with ethephon, the germination rate was only 76% (Figure 3). The pre-treatment
The germination
of seeds with ethephon percentage of M. faster
also promoted zehntneri
seedseeds using in with
germination, vitrothe conditions in a cul-of
greater number
ture medium
seeds was 74%
germinated within(Figure
11–14 3),days,
higher than observed
compared to the using
controlthe
(18same
days)light conditions
(Figure 4) and inthe
the previous experiment with Petri dishes and filter paper (61.3%).
highest GSI (2.93) compared to untreated (2.14) and the other plant growth regulators (BAP, Seed imbibition in an
ethephon
GA3 or BAP solution
+ GAas3 ) aadded
pre-treatment
to the cultureresulted
media in a(Figure
significant
5). and striking increase in the
percentage of in vitro germinated seeds of M. zehntneri. The best treatment and response
of seeds to ethephon was reported in the culture medium without phytoregulators, which
 resulted in 98% seeds germinated (Figure 3). In the control culture medium without the
pre-treatment with ethephon, the germination rate was only 76% (Figure 3). The pre-treat-
ment of seeds with ethephon also promoted faster seed germination, with the greater
number of seeds germinated within 11–14 days, compared to the control (18 days) (Figure
Plants 2023, 12, 4127 5 of 13
4) and the highest GSI (2.93) compared to untreated (2.14) and the other plant growth
regulators (BAP, GA3 or BAP + GA3) added to the culture media (Figure 5).

Figure 3.
Figure 3. Germination percentage
percentage of
ofM.
M.zehntneri
zehntneriseeds
seedssubjected
subjectedtotothe
thepre-treatment byby
pre-treatment soaking
soakingin
ethephon solution or water (control) solution and cultivated under culture media
in ethephon solution or water (control) solution and cultivated under culture media containingcontaining the
cytokinin
the 6-Benzylaminopurine
cytokinin 6-Benzylaminopurine(BAP) and/or
(BAP) Gibberellic
and/or AcidAcid
Gibberellic 3). Shapiro–Wilk normality test
(GA(GA
3 ). Shapiro–Wilk normality
= 0.857; Levene test for homoscedasticity = 0.7278. Coefficient of variation = 32.7%. F-values for phy-
test = 0.857; Levene test for homoscedasticity = 0.7278. Coefficient of variation = 32.7%. F-values
toregulators (1.58 ns), ethephon (11.37 **), and interaction (0.82 ns). Significant at 1% (**) probability.
for phytoregulators (1.58 ns), ethephon (11.37 **), and interaction (0.82 ns). Significant at 1% (**)
ns, non-significant. Uppercase letters indicate differences between the phytoregulators in the cul-
probability. ns, non-significant. Uppercase letters indicate differences between the phytoregulators in
Plants 2023, 12, x FOR PEER REVIEWture media and lowercase letters indicate differences between the pre-treatment of seeds6 with of 14
the culture or
Ethephon media and
water. lowercase
PGR-free letters indicatewith
+ pre-treatment differences between
water acts the pre-treatment of seeds with
as a control.
Ethephon or water. PGR-free + pre-treatment with water acts as a control.

(-ETHEPHON) (+ETHEPHON)
2.50
2.5
2.5
Average germination speed (AGS)

y = -0.0035x2 + 0.1684x - 0.0628

2.00 R² = 0.7331*
2.0

1.5
1.50

1.00
1.0
y = -0.0024x2 + 0.1201x - 0.1022
0.5
0.50 R² = 0.7543*

0.0
0.00
0 10 20 30 40 50
Time of germination (days)

Figure 4.
Figure 4. Average
Average germination
germination speed
speed (AGS)
(AGS) of
of M.
M. zehntneri
zehntneri seeds
seeds cultivated
cultivated under
under different
different wave-
wave-
lengths (blue, deep red, red and blue, white) sourced by LEDs and photosynthetically photon
lengths (blue, deep red, red and blue, white) sourced by LEDs and photosynthetically photon flux flux
densities (PPFDs). * Significant at 5% probability.
densities (PPFDs). * Significant at 5% probability.

3.12 a
index

2.93 A
 0 10 20 30 40 50
Time of germination (days)

Plants 2023, 12, 4127 Figure 4. Average germination speed (AGS) of M. zehntneri seeds cultivated under different 6wave-
of 13
lengths (blue, deep red, red and blue, white) sourced by LEDs and photosynthetically photon flux
densities (PPFDs). * Significant at 5% probability.

3.12 a

Germination speed index

2.93 A

2.48 ab 2.55 ab
2.14 B
1.98 b

CONTROL BAP GA3 BAP + GA3 WATER ETHEPHON

PHYTOREGULATORS PRE-TREATMENT

Figure5.
Figure 5. Germination
Germination Speed
Speed Index
Index (GSI)
(GSI) of M. zehntneri seeds subjected to different phytoregulators
different phytoregulators
added to
added to the
theculture
culturemedia
mediaandandpre-treatment
pre-treatmentwith
withethephon.
ethephon. Shapiro–Wilk
Shapiro–Wilk normality
normality test
test == 0.63;
0.63;
Levene test for homoscedasticity = 0.0975. Coefficient of variation = 28.3%. F-values for phytoregu-
Levene test for homoscedasticity = 0.0975. Coefficient of variation = 28.3%. F-values for phytoregula-
lators (2.75 ns), ethephon (9.98 **), and interaction (0.49 ns). Significant at 1% (**) probability. Low-
tors (2.75 ns), ethephon (9.98 **), and interaction (0.49 ns). Significant at 1% (**) probability. Lowercase
ercase letters indicate differences between the phytoregulators in the culture media and uppercase
letters indicate differences between the phytoregulators in the culture media and uppercase letters
letters indicate differences between the pre-treatment of seeds with Ethephon or water. ns, non-
indicate differences between the pre-treatment of seeds with Ethephon or water. ns, non-significant.
significant.
3. Discussion
3.1. Seed Dormancy in Cactaceae
Different authors reported dormancy in many Cactaceae species related to the xeric
environment, with highly limited natural resources to support seed germination, seedling
development, and survival. Some of these studies associate physical dormancy with
mechanical resistance [16,17] caused by the rigid coat surrounding the embryo, which
could limit the water uptake into the seeds. Seeds of different species of Cactaceae have
rigid and hard coats [18,19]. However, recent studies increased the evidence that only
physiological dormancy exists in Cactaceae seeds [20,21]. In Melocactus, the presence of
seed dormancy was widely reported, with percentages of germinated seeds between 8
and 65%, depending on the species, genotype, harvest time, germination temperature,
cultivation conditions (in vitro, Petri dish, or in vivo), and pre-treatments or treatments
applied to the seeds [22–24]. However, the main biological evidence and reasons why
35–90% of seeds did not germinate, and the treatments required to further increase the
germination of dormant seeds in Cactaceae, and more specifically in Melocactus, are not yet
fully elucidated.

3.2. Light Strongly Influenced Seed Germination in Melocactus zehntneri

Regarding the wavelengths, except for monochromatic blue light that reduced the
germination percentage of M. zehntneri seeds (38%), all other light wavelengths resulted in
germination percentages between 61.0% and 62.0% under filter paper conditions. Except
for monochromatic blue, all other LEDs tested in this study contained in their spectral
composition, emission peaks in the red wavelength range, such as the monochromatic
red, red/blue, and white LEDs. Red light appears to be the most important wavelength
that promotes, increases, and accelerates the germination of M. zehntneri seeds. Red
light is an important wavelength in mediating seed germination. The photomorphogenic
Plants 2023, 12, 4127 7 of 13

responses, which involve light receptors like phytochromes, control plant development
through the presence or absence of light and also the information and interpretation of
different wavelengths in the environment, which guide the most appropriate development,
including seed germination [25,26]. Cho et al. [27] also demonstrated that germination
of Arabidopsis seeds is promoted in red light-enriched environments by the activation of
Phytochrome B, resulting in a gradual increase in gibberellins that trigger germination.
Interestingly, different studies in Cactaceae species sought to correlate the presence of
light with increasing GA3 in seeds due to germination gains with these treatments [28].
However, in the present study, there was no strong evidence and no effectiveness of the
application of external GA3 . These results are in agreement with Barrios et al. [20] who
analyzed different studies and did not find strong evidence of an association between GA3
and dormancy releasing in Cactaceae seeds [20]. The percentage of germinated seeds under
different treatments rarely exceeds the average values observed in the present study with
M. zehntneri.
The monochromatic blue light substantially reduced seed germination in M. zehntneri.
The reduction in seed germination in response to blue light is not exclusive to the family
Cactaceae, and it is frequently reported to inhibit the germination of dormant seeds in
cultivated grasses [29,30]. Some studies suggest that the interaction of blue light with
cryptochrome 1 results in an increased concentration of Abscisic Acid (ABA) that inhibits
germination [31,32].
The majority (80.9%) from 275 studied Cactaceae species are considered positive
photoblastic and, any negative photoblastic was reported [20,21]. Flores et al. [33] reported
a strong influence of different Cactaceae tribes (phylogeny) on the response to light for
seed germination. This study showed that the most of the Cactaceae tribes have a strong
response to light for germination and that this differential response are due the size and
weight of the seeds presented in different tribes [20,33]. Flores et al. [34] reported that
from a total of 28 cactus species, all were considered positive photoblastic, and these
authors also described the occurrence of secondary dormancy as a consequence of seed
exposure to a period of darkness during germination. The same result was observed
with M. zehntneri in the present study, in which light was necessary for seed germination,
and exposure of seeds to dark conditions, even for short periods (10, 20, and 30 DAS),
significantly reduced the seed germination percentage (Table 1). Interestingly, even after
a short period of darkness (10 days), seeds did not germinate even after up to 12 months
under the same light conditions that promote germination, with the probable acquisition of
secondary dormancy due to the absence of light. Under light conditions, the percentage of
germinated seeds of M. zehntneri was, on average, 40–60% [15], showing their photoblastic
positive response.

3.3. Ethephon Releases the Germination of Dormant Seeds of M. zehntneri

Interestingly, the in vitro seeding and germination increased the germination per-
centage of seeds (76% in control) of M. zehntneri compared to the previous experiments
in filter paper (≡60% germination) and showed that in vitro conditions can be used for
this cactus species. The main differences between these two experiments explain that
these differences are the result of the use of a culture media (nutrients, sucrose, and other
components) instead of deionized water under filter paper conditions, as well as the use of
pre-treatment with water for 24 h before seeding instead of only 10 min, which was used
for the experiment with filter paper and previously reported in another study [15].
In our study, we used the in vitro conditions to test the effect of phytoregulators on
the germination of this cactus. Treatments with phytoregulators have been used to break
dormancy in seeds of different Cactaceae species. The most important effects observed in
the present study with M. zehntneri occurred when seeds under in vitro conditions were pre-
treated with ethephon (2-chloroethylphosphonic acid), showing a germination percentage
of 98% of seeds, compared to 76% with water pre-treatment used as a control. In this way,
ethephon was the unique treatment promoting the germination of dormant seeds in M.
Plants 2023, 12, 4127 8 of 13

zehntneri, but the knowledge about how this phytoregulator affects cactus development is
still limited. Among the few reports, ethylene is responsible for the closing, wilting, and
pollination of flowers [35] and fruit ripening [36] in some cactus species. However, the
effects of ethylene on releasing seed dormancy in Cactaceae have not yet been reported.
Although little explored and used in Cactaceae, ethylene is considered a key hormone
that regulates dormancy and seed germination, as well as the establishment of seedlings
after germination in many plant species. This hormone is effective, at concentrations from
0.1 to 200 µL L−1 , in releasing seeds from dormancy [13]. In the present study, the pre-
treatment with ethephon was effective in releasing the germination of M. zehntneri seeds
when applied by immersing seeds in a solution at 100 µL L−1 for 24 h using the commercial
product Ethrel® (Bayer® , Belford Roxo, Brasil), which contains 240 g L−1 ethephon, thus,
at a concentration of 24 µL L−1 ethephon. Ethylene acts in the release of seeds from
dormancy, especially involving a complex interaction with other hormonal groups in seeds,
such as ABA, Gibberellins, Nitrous Oxide (NO), and reactive oxygen species (ROS) [37].
Different studies have demonstrated that, through the use of ethylene biosynthesis and
action inhibitors, ET insensitive mutants, and the use of ET biosynthesis precursors, such
as 1-aminocyclopropane 1-carboxylic acid (ACC), ethylene is involved in overcoming the
dormancy and promoting germination [13]. In addition, this hormone can also neutralize
the effects of ABA on seed dormancy.
Among the phytoregulators used to study germination, one of the most used was the
GA3 [38]. This phytoregulator has shown divergent results in releasing dormant seeds of
Cactaceae, in some cases even reducing the percentage of germinated seeds, as observed
in Ferocactus species [39]. In the present study, seeds of M. zehntneri did not respond to
the presence of GA3 at 1.0 mg L−1 in the culture medium (72% germinated seeds) and
did not differ from the control without phytoregulators (76% germinated seeds). These
results diverge from the in vitro germination of M. sergipensis, in which the addition of
GA3 at 2 mg L−1 for 6 h increased the percentage of seeds germinated in this species.
However, the maximum percentage of in vitro germinated seeds observed for this species
was 38% [22], much lower than observed in our study with M. zehntneri. Nevertheless, the
limited positive results in increased germination by using GA3 in several cacti species may
be related to the differential sensitivity of seed tissues to GA3 [40].
Cytokinins and ethylene have been identified as hormonal groups that upregulate
themselves and help to overcome seed dormancy [41]. However, in the present study,
the addition of BAP, a synthetic cytokinin, or the BAP + GA3 combination in the culture
medium reduced the effects associated with germination promoted by the pre-treatment
containing ethephon. For example, in treatments containing this cytokinin in the culture
medium, there were no differences in the percentage of germinated seeds pre-treated or
not with ethephon. The effects of cytokinins on the germination of Cactaceae seeds are
practically non-existent but positive effects of cytokinins on increasing the germination
percentage and GSI have been demonstrated in species from other plant families [42,43].

4. Material and Methods

4.1. Plant Material
For the experiment, Melocactus zehntneri was used, from the germplasm collection
of the Center of Agrarian Sciences, UFSCar, catalog number ABBC280 (Figure 6) by the
Sistema Nacional de Gestão do Patrimônio Genético e Conhecimento Tradicional Associado
(SisGen/Brazil). Three adult plants in full fruiting were used as a source of seeds, and fruit
was collected at the moment they were detached from the cephalium (Figure 6).
 4.1. Plant Material
For the experiment, Melocactus zehntneri was used, from the germplasm collection of
the Center of Agrarian Sciences, UFSCar, catalog number ABBC280 (Figure 6) by the
Sistema Nacional de Gestão do Patrimônio Genético e Conhecimento Tradicional Associ-
Plants 2023, 12, 4127 9 of 13
ado (SisGen/Brazil). Three adult plants in full fruiting were used as a source of seeds, and
fruit was collected at the moment they were detached from the cephalium (Figure 6).

Figure
Figure6.6.Adult
Adultplant of of
plant M.M.zehntneri with
zehntneri cephalium
with (left)
cephalium and and
(left) details of cephalium
details containing
of cephalium ma-
containing
ture
mature fruit (right) used to collect seeds for the experimental procedures. Original photos ofand
fruit (right) used to collect seeds for the experimental procedures. Original photos of JCC JCC
MFCM.
and MFCM.

4.2.Seed
4.2. SeedPreparation
Preparationand
andStorage
Storage
Seedswere
Seeds wereremoved
removedfrom fromthethe fruit
fruit andand washed
washed in ain a sieve
sieve under under running
running waterwater
con-
containing
taining a fewadrops
few drops of neutral
of neutral detergent
detergent to remove
to remove excessexcess
mucilagemucilage surrounding
surrounding the
the seeds.
seeds.washing,
After After washing, seeds
seeds were were in
placed placed
a growin aroom
growatroom
25 °C–28 °C to◦ C
at 25–28 to on
dry dryfilter
on filter paper
paper for
forh.24
24 h. Seeds
Seeds remained
remained stored
stored that way
that way for another
for another 14 days
14 days until until the experiments.
the experiments. This
This pro-
cedure of removing mucilage and drying, demonstrated by previous experiments, is theis
procedure of removing mucilage and drying, demonstrated by previous experiments,
the best
best condition
condition forgermination
for the the germination of seeds
of seeds of species.
of this this species.

4.3.Experiment
4.3. Experimentunder
underDifferent
DifferentLight
LightIntensities
Intensitiesand
andWavelengths
Wavelengthsfor
forGermination
Germination
Inthe
In the implementation
implementationof ofthe
theexperiment,
experiment,seeds
seedsof M. zehntneri
ofM. zehntneriwere
were immersed
immersedinin
distilled water for 10 min, according to results previously obtained by our laboratory
distilled water for 10 min, according to results previously obtained by our laboratory team
team [14]. Later, using a laminar flow cabinet, seeds were subjected to asepsis, aiming
[14]. Later, using a laminar flow cabinet, seeds were subjected to asepsis, aiming at the
at the reduction or even elimination of microorganisms. This was performed in 15 mL
Falcon® tubes and seeds were immersed in 70% alcohol (v/v) for one minute and then
in a solution containing 30% sodium hypochlorite (2.0–2.5% active chlorine) added with
5 drops of neutral detergent for every 100 mL solution for 15 min under constant stirring,
followed by three washes in previously autoclaved deionized water. In the last rinse,
approximately 2 mL of deionized water with pH adjusted to 5.8 was kept as a vehicle for
seed inoculation on the plates. All seeds, 20 per plate, were sown in clear, smooth, and
sterile polystyrene Petri dishes, previously filled with two layers of filter paper saturated
with 5 mL sterile deionized water at pH 5.8 per dish. This germination system was called
filter paper condition. Subsequently, dishes containing the seeds were arranged under
different light intensities (I, II, and III) and wavelengths given by LED lamps and cultivated
in a grow room, as follows: blue LED (Phillips Greenpower LED Research Module Blue,
~440 nm, Pila, Poland), red LED (Phillips Greenpower LED module HF deep red, ~660 nm,
Pila, Poland), blue (1)/red (1.5) LED (LabPar, with wavelength peaks at 447 nm, range
420–470 nm—blue and 667 nm, range 625–680 nm—red, Barueri, Brazil), and as a white
LED control (Ourolux® , São Paulo, Brazil), with peaks at 440–450 nm (blue), 540–550 nm
(green) and 610–620 nm (red).
Photosynthetically photon flux densities (PPFD) were measured using a PPFD Quan-
tum meter, Apogee Instruments® , Model SQ-520 (North Logan, UT, USA) of each light
source and were obtained by placing the dishes in equidistant distances from the LEDs
(Table 2).
Plants 2023, 12, 4127 10 of 13

Table 2. Photosynthetically Photon Flux Density (PPFD) sourced by LED wavelengths used for seed
germination in Melocactus zehntneri. I, II, and III refer to the treatments using different light intensities
where seeds were cultivated.

Photosynthetically Photon Flux Density

(µmol m−2 s−1 )
LED
I II III Mean
Wavelength
Blue 37.83 ± 0.50 67.19 ± 0.42 134.21 ± 0.54 79.74
Deep Red 53.59 ± 0.64 97.58 ± 0.73 144.89 ± 0.70 98.69
Blue/Red LED 41.28 ± 0.33 64.02 ± 0.75 115.19 ± 0.78 73.50
White LED 56.83 ± 0.59 96.19 ± 0.21 135.57 ± 0.36 96.20
Mean 47.38 81.25 132.47

The experiment was a completely randomized design in a 3 × 4 factorial arrangement

(PPFD × wavelength) with four replications composed of individual Petri dishes containing
20 seeds each. As a complementary experiment, dark time influence on the germination
of M. zehntneri seeds was tested, in which seeds were kept protected from light in a grow
room for three periods: 10, 20, or 30 days of darkness. In this case, seeds under germination
conditions were only exposed to a light source after remaining in the respective periods
in the dark. For this, the same procedure of preparation and inoculation of seeds was
carried out, and the experimental design was completely randomized. Petri dishes from
both experiments were sealed with a transparent PVC film and kept in a grow room at
26 ± 1 ◦ C and a photoperiod of 14 h. Germination was evaluated twice a week. Seeds were
considered germinated when the hypocotyl-radicle protrusion was equal to or greater than
0.1 cm. In the end, the Germination Percentage (G%), Average Germination Speed (AGS),
and Germination Speed Index (GSI) were calculated.

4.4. In Vitro Germination of M. zehntneri under Different Concentrations of Phytoregulators

The main objectives of this experiment were to evaluate the effect of different classes
of phytoregulators on the germination of M. zehntneri seeds and to develop a methodology
for in vitro germination as an alternative to the method of seed germination in Petri dishes.
Procedures before seeding and aiming at seed asepsis were carried out as described in
the previous experiment. In the last rinse, approximately 2 mL deionized water (pH~5.8)
was maintained so that this solution containing the seeds could be used as a vehicle
for inoculation in culture flasks containing 30 mL MS culture medium [44], containing
sucrose (20 g L−1 ), inositol (100 mg L−1 ), activated charcoal (1 g L−1 ), and the pH was
adjusted to 5.8 before the addition of agar (6.4 g L−1 ). Flasks containing the culture
medium were autoclaved at 120 ◦ C for 25 min. Phytoregulators tested for germination
of M. zehntneri seeds and added to the MS culture medium were 6-benzylaminopurine
(BAP) 4.44 µM; gibberellic acid (GA3 ) 2.89 µM; and the combination of the two, BAP
(4.44 µM) add GA3 (2.89 µM), and control without addition of phytoregulators. Also, we
evaluated the effect of pre-treatment of seeds in a solution containing 100 µL L−1 Ethrel®
(240 g/L ethephon—Bayer® , Belford Roxo, Brazil, totaling a concentration of 166 µM) for
24 h before the experiment and later inoculated in culture media containing the same
treatments described above. After inoculation, flasks containing the seeds were kept in a
grow room under the same conditions as in the previous experiment but using only the
LED in the blue (1) and red (1.5) wavelengths.
The experiment was conducted in a Completely Randomized Design (CRD), in a 2 (pre-
treatment with Ethrel® ) × 4 (phytoregulators in the culture medium) factorial arrangement.
In total, six replications were performed per treatment; each replication consisted of a flask
containing 30 mL culture medium, with ten seeds per flask. Germination was checked
twice a week, and seeds were only considered germinated when embryo protrusion was
equal to or greater than 0.1 cm. After 42 days, the germination percentage (%G), the
Plants 2023, 12, 4127 11 of 13

AGS, and GSI were also evaluated. Germination Percentage (G%) was determined by
G% = ∑_ni/N × 100, where ∑ni is the total number of germinated seeds, and N is the total
number of seeds tested [45]. The Average Germination Speed was determined according
to the expression AGS = (ni)/ti, in which “ni” is the number of seeds germinated at time
“i” and “ti” is the time after implementation of the test [46]. The Germination Speed Index
(GSI) was determined using the equation GSI = G_1/N_1 + G_2/N_2 . . . + G_n/N_n,
where G1, G2, . . . Gn refer to the number of germinated seeds and N1, N2, Nn, to the
number of days after seeding [47].

4.5. Statistical Analysis

Data obtained were tested by analysis of variance (ANOVA). For the comparison of
means, Tukey’s test was applied at 5% significance, using the software AgroEstat v1.1 [48]
and RStudio v.3.1.1 [49].

5. Conclusions
An effective protocol was developed, which achieved high percentages of germina-
tion in M. zehntneri. Red light promoted and accelerated the germination of seeds of M.
zehntneri, while monochromatic blue light reduced the germination percentage in this
species. The increase of PPFDs of light is positively correlated with the germination of
cactus seeds. The dark conditions during germination for short periods drastically reduced
seed germination. Ethephon used as pre-treatment played a key role in the M. zehntneri
seed germination (98%), releasing the germination of dormant seeds. The present study
can serve as a reference for studies aiming to overcome seed dormancy in Melocactus, and
in vitro germination can still serve as a tool for studying the germination and propagation
of cacti species, aiming at their large-scale production or even for conservation purposes.
Studies are required to increase knowledge of the effects of ethylene on the germination
of seeds of other cactus species, as well as its mode of action in overcoming dormancy
in Cactaceae.

Author Contributions: Conceptualization, J.C.C. and M.F.C.M.; methodology, J.C.C. and M.F.C.M.;
validation, J.C.C.; formal analysis, M.F.C.M. and J.C.C.; investigation, M.F.C.M. and J.C.C.; resources,
J.C.C.; data curation, M.F.C.M. and J.C.C.; writing—original draft preparation, J.C.C. and M.F.C.M.;
writing—review and editing, J.C.C.; supervision, J.C.C.; project administration, M.F.C.M. and J.C.C.;
funding acquisition, M.F.C.M. and J.C.C. All authors have read and agreed to the published version
of the manuscript.
Funding: This study was supported by the Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP, Brazil) (Process number 2021/01814-8) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPQ, Brazil) (Process number 311083/2018-8).
Data Availability Statement: The data could be shared if required.
Acknowledgments: The authors thank Willian Naves Duarte and Matheus Armelin Nogueira for
their intellectual contributions to this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
BAP, Benzylaminopurine; Ethephon, 2-chloroethylphosphonic acid; GA3 , Gibberellic acid; GSI, Ger-
mination speed index; AGS, Average germination speed; LED, Light-emitting diodes; MS, Murashige
and Skoog medium; PPFD, Photosynthetically Photon Flux Density.

References
1. Machado, M. The genus Melocactus in eastern Brazil: Part I—An introduction to Melocactus. Br. Cactus Succul. J. 2009, 27, 5–20.
2. Cavalcante, A.M.; Vasconcelos, G.C.L. Comércio legal de cactos ornamentais: Oportunidade para uso sustentável no semi-árido
do brasil. Rev. Econ. Nord. 2016, 47, 9–19. [CrossRef]
Plants 2023, 12, 4127 12 of 13

3. Martinelli, G.; Moraes, M.A. Livro Vermelho da Flora do Brasil; Centro Nacional de Conservação da Flora—CNCFlora; Pesquisas
Jardim Botânico do Rio de Janeiro: Rio de Janeiro, Brazil, 2013.
4. Cites. Criteria for Amendment of Appendices I and II. Resolution Conf. 9.24, 1994, (Rev. CoP17). Available online: https:
//cites.org/sites/default/files/document/E-Res-09-24-R17.pdf (accessed on 6 December 2023).
5. Cites. Trade Database. 2019. Available online: https://trade.cites.org (accessed on 16 May 2022).
6. Filho, E.S.B.; de Santana, M.C.; Santos, P.A.A.; Ribeiro, A.d.S. Levantamento etnobotânico da família Cactaceae no estado de
Sergipe. Rev. Fitos 2018, 12, 41–53. [CrossRef]
7. Machado, M.; Taylor, N.P.; Braun, P.; Zappi, D. Melocactus Zehntneri. The IUCN Red List of Threatened Species. 2017. Available
online: https://www.iucnredlist.org/species/152094/121521664 (accessed on 19 June 2022).
8. Fabricante, J.R.; Andrade, L.A.; Marques, F.J. Caracterização populacional de Melocactus zehntneri (Britton & Rose) Luetzelburg
(Cactaceae) ocorrente em um inselbergue da Caatinga paraibana. Biotemas 2010, 23, 61–67. [CrossRef]
9. Zappi, D.; Silva, S.; Taylor, N.; Machado, M. Plano de Ação Nacional Para a Conservação das Cactáceas; N◦ 24; Instituto Chico Mendes
de Conservação da Biodiversidade, ICMBIO: Cabedelo, Brazil, 2011; pp. 30–52.
10. Correia, D.; Nascimento, E.H.; Santiago, L.G.; Filho, A.A.; Morais, J.P. Germinação In Vitro de Sementes de Coroa-de-Frade (Melocactus
sp.); Embrapa Agroindústria Tropical: Fortaleza, Brazil, 2018; pp. 6–15.
11. Lafite, N.F.; Salimon, C.I. Melocactus violaceus Pfeiff. (Cactaceae) growth rate and abundance in a sandy terrace of Atlantic Rain
Forest (REBIO Guaribas, Paraíba, Brazil). Pesqui. Ensino Ciências Exatas Nat. 2020, 4, 1–9. [CrossRef]
12. Bárbara, E.P.; Silva, A.A.; Souza, M.M.; Gurgel, Z.E.; Marchi, M.N.; Bellintani, M.C. Germinação e Criopreservação de Sementes
de Cactos Nativos da Bahia. Gaia Sci. 2015, 9, 91–96.
13. Corbineau, F.; Xia, Q.; Bailly, C.; El-Maarouf-Bouteau, H. Ethylene, a key factor in the regulation of seed dormancy. Front. Plant
Sci. 2014, 5, 539. [CrossRef]
14. Santos, L.R. Desenvolvimento de Protocolo de Propagação de Espécies de Melocactus em Situação de Risco de Extinção. Master’s
Thesis, Universidade Federal de Sergipe, São Cristóvão, Brazil, 2019.
15. Magnani, M.F.C.; Cardoso, J.C. Seed germination and substrates for seedlings cultivation of Melocactus zehntneri. Hortic. Bras.
2022, 40, 384–392. [CrossRef]
16. Rojas-Aréchiga, M.; Aguilar, K.M.; Golubov, J.; Mandujano, M.C. Effect of Gibberellic Acid on Germination of Seeds of Five
Species of Cacti From the Chihuahuan Desert, Northern Mexico. Southwest. Nat. 2011, 56, 393–400. [CrossRef]
17. Pérez-Molphe-Balch, E.; Santos-Díaz, M.d.S.; Ramírez-Malagón, R.; Ochoa-Alejo, N. Tissue culture of ornamental cacti. Sci.
Agricola 2015, 72, 540–561. [CrossRef]
18. Archibald, E.E. The development of the ovule and seeds of jointed cactus (Opuntia aurantiaca, Lindley). S. Afr. J. Sci. 1939, 36,
195–211.
19. Orozco-Segovia, A.; Marquez-Guzman, J.; Sanchez-Coronado, M.E.; de Buen, A.G.; Baskin, J.M.; Baskin, C.C. Seed Anatomy
and Water Uptake in Relation to Seed Dormancy in Opuntia tomentosa (Cactaceae, Opuntioideae). Ann. Bot. 2007, 99, 581–592.
[CrossRef]
20. Barrios, D.; Sánchez, J.A.; Flores, J.; Jurado, E. Seed traits and germination in the Cactaceae family: A review across Americas. Bot.
Sci. 2020, 98, 417–440. [CrossRef]
21. Rojas-Aréchiga, M.; García-Morales, E. Dormancy and viability of Ferocactus peninsulae (Cactaceae) seeds. Plant Species Biol. 2022,
37, 173–181. [CrossRef]
22. Zamith, L.R.; Cruz, D.D.; Richers, B.T. The effect of temperature on the germination of Melocactus violaceus Pfeiff. (Cactaceae), a
threatened species in restinga sandy coastal plain of Brazil. An. Acad. Bras. Ciênc. 2013, 85, 615–622. [CrossRef]
23. Filho, E.S.B.; de Santana, M.C.; Santos, P.A.A.; de Souza Ribeiro, A. Germinação e aclimatização de Melocactus sergipensis Taylor &
Meiado. Iheringia Série Botânica 2019, 74, e2019009. [CrossRef]
24. Chaves, V.T.; Santos, E.P.; Rodrigues, A.C. Medium MS at different concentrations in the initial development in vitro of Melo-
cactus conoideus Buining & Brederoo. Rev. Scientia 2021, 6, 81–91.
25. Kami, C.; Lorrain, S.; Hornitschek, P.; Fankhauser, C. Light-Regulated Plant Growth and Development. Curr. Top. Dev. Biol. 2010,
91, 29–66.
26. Neff, M.M.; Sanderson, L.; Tedor, D. Light-Mediated Germination in Lettuce Seeds: Resurrection of a Classic Plant Physiology
Lab Exercise. Am. Biol. Teach. 2009, 71, 367–370. [CrossRef]
27. Cho, Y.-H.; Hong, J.-W.; Kim, E.-C.; Yoo, S.-D. Regulatory Functions of SnRK1 in Stress-Responsive Gene Expression and in Plant
Growth and Development. Plant Physiol. 2012, 158, 1955–1964. [CrossRef]
28. Ortega-Baes, P.; Rojas-Aréchiga, M. Seed germination of Trichocereus terscheckii (Cactaceae): Light, temperature and gibberellic
acid effects. J. Arid. Environ. 2007, 69, 169–176. [CrossRef]
29. Hoang, H.H.; Sechet, J.; Bailly, C.; Leymarie, J.; Corbineau, F. Inhibition of germination of dormant barley (Hordeum vulgare L.)
grains by blue light as related to oxygen and hormonal regulation. Plant Cell Environ. 2014, 37, 1393–1403. [CrossRef]
30. Jacobsen, J.V.; Barrero, J.M.; Hughes, T.; Julkowska, M.; Taylor, J.M.; Xu, Q.; Gubler, F. Roles for blue light, jasmonate and nitric
oxide in the regulation of dormancy and germination in wheat grain (Triticum aestivum L.). Planta 2013, 238, 121–138. [CrossRef]
31. Barrero, J.M.; Downie, A.B.; Xu, Q.; Gubler, F. A Role for Barley CRYPTOCHROME1 in Light Regulation of Grain Dormancy and
Germination. Plant Cell 2014, 26, 1094–1104. [CrossRef]
Plants 2023, 12, 4127 13 of 13

32. Hofmann, N. Cryptochromes and Seed Dormancy: The Molecular Mechanism of Blue Light Inhibition of Grain Germination.
Plant Cell 2014, 26, 846. [CrossRef]
33. Flores, J.; Jurado, E.; Chapa-Vargas, L.; Ceroni-Stuva, A.; Dávila-Aranda, P.; Galíndez, G.; Gurvich, D.; León-Lobos, P.; Ordóñez,
C.; Ortega-Baes, P.; et al. Seeds photoblastism and its relationship with some plant traits in 136 cacti taxa. Environ. Exp. Bot. 2011,
71, 79–88. [CrossRef]
34. Flores, J.; Jurado, E.; Arredondo, A. Effect of light on germination of seeds of Cactaceae from the Chihuahuan Desert, Mexico.
Seed Sci. Res. 2006, 16, 149–155. [CrossRef]
35. Van Doorn, W.G. Does Ethylene Treatment Mimic the Effects of Pollination on Floral Lifespan and Attractiveness? Ann. Bot. 2002,
89, 375–383. [CrossRef]
36. Esparza, F.; Esparza, G.; Macias, F.J.; Méndez, S.J. Effect of Ethephon on ripening of cactus pear fruits. Acta Hortic. 2006, 728,
165–172. [CrossRef]
37. Arc, E.; Sechet, J.; Corbineau, F.; Rajjou, L.; Marion-Poll, A. ABA crosstalk with ethylene and nitric oxide in seed dormancy and
germination. Front. Plant Sci. 2013, 4, 63. [CrossRef]
38. Mascot-Gómez, E.; Flores, J.; López-Lozano, N.E.; Yáñez-Espinosa, L. Seed germination of Southern Chihuahuan desert cacti:
Effect of mucilage, light and phytohormones. Flora 2020, 263, 151528. [CrossRef]
39. Amador-Alférez, K.A.; Díaz-González, J.; Loza-Cornejo, S.; Bivián-Castro, E. Efecto de diferentes reguladores de crecimiento
vegetal sobre la germinación de semillas y desarrollo de plántulas de dos especies de Ferocactus (Cactaceae). Polibotánica 2013, 35,
109–131.
40. Kucera, B.; Cohn, M.A.; Leubner-Metzger, G. Plant hormone interactions during seed dormancy release and germination. Seed
Sci. Res. 2005, 15, 281–307. [CrossRef]
41. Zdarska, M.; Dobisová, T.; Gelová, Z.; Pernisová, M.; Dabravolski, S.; Hejátko, J. Illuminating light, cytokinin, and ethylene
signalling crosstalk in plant development. J. Exp. Bot. 2015, 66, 4913–4931. [CrossRef]
42. Nikolić, R.; Mitić, N.; Miletić, R. Effects of Cytokinins on In Vitro Seed Germination and Early Seedling Morphogenesis in Lotus
corniculatus L. J. Plant Growth Regul. 2006, 25, 187–194. [CrossRef]
43. Wang, Y.; Li, L.; Ye, T.; Zhao, S.; Liu, Z.; Feng, Y.Q.; Wu, Y. Cytokinin antagonizes ABA suppression to seed germination of
Arabidopsis by downregulating ABI5 expression. Plant J. 2011, 68, 249–261. [CrossRef]
44. Murashige, T.; Skoog, F. A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures. Physiol. Plant. 1962,
15, 473–497. [CrossRef]
45. Labouriau, L.G. A Germinação das Sementes. Monografias Científicas; Organização dos Estados Americanos: Washington, DC, USA,
1983; p. 170.
46. Carvalho, D.B.; Carvalho, R.I. Qualidade fisiológica de sementes de guanxuma em influência do envelhecimento acelerado e da
luz envelhecimento acelerado e da luz. Acta Sci. Agron. 2009, 31, 489–494. [CrossRef]
47. Maguire, J.D. Speed of Germination—Aid In Selection And Evaluation for Seedling Emergence And Vigor. Crop Sci. 1962, 1,
176–177. [CrossRef]
48. Barbosa, J.C.; Maldonado Júnior, W. Software AgroEstat: Sistema de Análises Estatísticas de Ensaios Agronômicos; Universidade
Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias: Jaboticabal, Brasil, 2009.
49. Solanki, H.; Patel, Y.; Vaghela, D.; Vaidya, M. R: An Open Source Software Environment for Statistical Analysis. Inflibnet 2017, 24,
14–21.

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