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Citation: Bertsouklis, K.; Panagaki,
K.-P. In Vitro Germination and
Propagation of Dyckia brevifolia, An
Ornamental and Endangered
Bromeliad. Horticulturae 2022,8, 390.
https://doi.org/10.3390/
horticulturae8050390
Academic Editor: Sergio
Ruffo Roberto
Received: 30 March 2022
Accepted: 25 April 2022
Published: 28 April 2022
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horticulturae
Article
In Vitro Germination and Propagation of Dyckia brevifolia, An
Ornamental and Endangered Bromeliad
Konstantinos Bertsouklis * and Konstantina-Panagiota Panagaki
Laboratory of Floriculture and Landscape Architecture, Department of Crop Science, School of Plant Sciences,
Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece; panagakitina@gmail.com
*Correspondence: kber@aua.gr; Tel.: +30-210-5294558
Abstract:
Dyckia brevifolia is an endangered plant used for ornamental purposes. As no references to
the
in vitro
propagation of the species exist, the present study aims at investigating the possibility
of an efficient micropropagation protocol. Seeds collected from mother plants were germinated at
high percentages (84–86%) at a range of 15–25
◦
C, without any pre-treatment, and demonstrated
their highest germination speed index (191.51) at 25
◦
C.
In vitro
-grown seedlings were used as the
starting material for micropropagation on solid, or liquid, MS medium, supplemented with a variety
of concentrations of cytokinins (BA, KIN or 2IP). Shoots and leaves were used as starting explants.
Liquid media supplemented with BA or 2IP at 1.0 mg L
−1
led to high multiplication rate and 2.7,
or 2.3, lateral shoots were regenerated while on 2IP a high percentage (77.5%) of rooting occurred
at the same time. Rooted microshoots were acclimatised ex vitro at 100% and acclimatised plants
were transplanted in pots where they grew with a survival rate of 100% after two months. The
in vitro
propagation protocol presented in this study could enhance the large-scale propagation use
of D. brevifolia as an ornamental plant and, simultaneously, contribute to the ex-situ conservation of
the species.
Keywords: acclimatisation; Bromeliaceae; GSI; liquid culture; micropropagation; tissue culture
1. Introduction
Bromeliads are widely used in floriculture. Their attractive rosettes, range of colours
of flowers, and graceful foliage, make them ideal ornamental plants. Still, information on
their reproductive strategies remains scant [
1
,
2
]. Certain members of Bromeliaceae are often
viewed as a prime example of adaptive radiation [
3
] as they can be found in a wide range
of environments, from mesic to xeric, from terrestrial to epiphytic, and from sea level areas
to high elevation ones. Their key adaptations include leaf succulence, spiny leaves, which
gives them herbivore protection, and a crassulacean acid metabolism (CAM), which allows
them to inhabit rocky, semi-arid environments [
4
,
5
]. However, bromeliads are relatively
difficult to propagate, with one plant producing no more than a few individual plantlets
characterised by slow growth [
6
]. Thus, in the recent past, the development of plant tissue
culture-based techniques has been gaining ground since such techniques have proved
valuable tools in developing new strategies for the bromeliads’ mass propagation [7].
Dyckia is a genus that includes 170 species (either terrestrial or saxicolous, tankless
perennial plants) and is one of the most species-rich genera in the Pitcairnioideae sub-
family [
8
]. Many of the Dyckia members are of high ornamental interest [
9
]. According
to recent studies, the Dyckia genus is one of the three Bromeliaceae genera which have
formed a well-supported xerophytic clade [
4
,
10
,
11
], and includes species which are native
to and thrive in environments with poor soil, limited water supply, and high sunlight
exposure [
12
]. Dyckia brevifolia Baker (aka ‘sawblade’) is a species endemic to Southern
Brazil [
13
]. It has yellow flowers on a 30-cm-long spike. It forms about 30 leaves, with
fierce spiny margins (20 cm long), grouped in a dense rosette. Leaves are erect in the centre
Horticulturae 2022,8, 390. https://doi.org/10.3390/horticulturae8050390 https://www.mdpi.com/journal/horticulturae
Horticulturae 2022,8, 390 2 of 14
and recurve outwards, growing to a height of 40–50 cm [
14
]. D. brevifolia is a heliophyte
that tolerates full sunlight, but can also adapt to a river’s flow, whether such adaptation
entails its submergence during floods or its dehydration during periods of low tide [
15
].
What is more, the morphoanatomical aspects of D. brevifolia carry both xeromorphic and
hydromorphic characteristics which, in a rheophytic environment, give rise to adaptations
during periods of both low and high water [
16
]. At present, the species, a member of the
Brazilian consolidated ornamentals [
17
], can be found in an area no broader than a mere
two hectares [1] and is faced with extinction as a result of the human activities [13] taking
place in the Itajaí-Açu River Basin for construction of a hydroelectric plant [
18
]. It is similar
to Dyckia distachya, another rare bromeliad included in the official list of Brazilian species
threatened with extinction [
19
,
20
]. The special characteristics described above add such
a high ornamental value to the species that a study of seed germination, in tandem with
the development of
in vitro
propagation methods, could enhance its exploitation as an
ornamental plant and lead to the establishment of strategies for its conservation in situ and
ex situ alike.
In Greece, D.brevifolia is used as a landscape or potted plant, suitable for landscape
architecture. It can also be used as a xerophyte.
In vitro
propagation of the bromeliad can
take place using explants derived from the leaves’ basal region since application of plant
growth regulators can activate the vascular elements present in the explants [
6
]. This type of
explant has been successfully used in other bromeliad
in vitro
systems [
21
–
24
]. For instance,
in the case of Dyckia macedoi [
9
],
in vitro
morphogenesis was carried out starting from leaf
explants and, in the case of Dyckia distachya,
in vitro
morphogenesis began with flower
stalk segments [
7
]. In the cases of Dyckia sulphurea Koch [
25
] and Dyckia maritima Baker [
26
],
the role of starting material towards the establishment of a successful micropropagation
technique was played by shoot explants. Pompelli [
27
] as well as Pompelli and Guerra [
28
]
used seeds as starting material for
in vitro
propagation of Dyckia distachya Hassler and so
did Silva et al. [
29
] for Dyckia agudensis. The use of explants of seedling origin for
in vitro
cultures initiation does encourage a high proliferation rate [
30
], but the fact that Bromeliad
seeds lose their viability quite rapidly [
31
] must be taken into account. Propagation by
seed enhances genetic diversity which, in turn, contributes to the selection of genotypes of
exceptional commercial value [
32
–
34
], has little environmental impact [
35
], and is followed
by efficient clonal propagation strategies necessary in meeting the increased demands of
the ornamental plant market. Proper molecular methods should be employed, in tandem
with the study of the genetic variability in natural populations, for the assessment of
genetic diversity and the relationship between individuals aiming at the preservation of
natural variation. Cases of ex vitro seed germination of D. brevifolia have indeed been
reported [
36
,
37
]. However, to the best of our knowledge, there are no studies researching
in vitro propagation of D. brevifolia.
All Dyckia species show promising potential for the ornamental plant market [
37
].
Moreover, an efficient protocol for seed and clonal propagation could facilitate the introduc-
tion of suitable clones of D. brevifolia into nursery production and supporting floriculture
use. The use of seedlings as starting plant material has proved quite effective for other
species [
30
,
38
], enhancing those species’ high genetic diversity and making it possible
to select clones suitable for the floricultural industry as well as for breeding programs.
The present study examines the
in vitro
germinability of D. brevifolia seeds and the use of
in vitro
-grown seedlings as starting material in order to investigate the effect of explant
type, cytokinins, and medium phase of in vitro morphogenesis and micro-shoot rooting.
2. Materials and Methods
2.1. Plant Material
Seeds were collected fully mature, at Gryllis Water Lilies Nurseries (Marathon, Attica,
Greece, 38
◦
08
0
02.7
00
N 23
◦
56
0
33.7
00
E) from potted mother plants. They were transferred
to the Laboratory of Floriculture and Landscape Architecture, Agricultural University of
Athens, Athens. They were stored dry, in the dark, in 9-cm, unsealed, plastic Petri dishes
Horticulturae 2022,8, 390 3 of 14
between filter paper, at T = 25
◦
C, and a relative humidity of 30%. After three months of
collection, in-vitro germination and propagation took place (Figure 1).
Figure 1.
Schematic representation of Dyckia brevifolia
in vitro
propagation procedures starting from
in vitro germinated seeds.
2.2. In Vitro Germination
Before initiating germination treatments, seeds were surface-sterilized with 20% (v/v)
commercial bleach (4.6% w/v sodium hypochlorite) for 10 min and rinsed three times for
three min per time with sterile distilled water. Seeds were sown in 9-cm plastic Petri dishes
containing Hf, half-strength Murashige and Skoog (MS) medium [
39
]. Petri dishes were
incubated at 5, 10, 15, 20, 25, 30, and 35
◦
C. In compliance with the rules of the International
Seed Testing Association [
40
], germination was defined as the appearance of a radicle that
would be at least 2 mm long and would be recorded every 1 d. T
50
was defined as time
taken by cumulative germination to reach 50% of its maximum [
41
]. One hundred seeds
were used per treatment (five Petri dishes per treatment/20 seeds per Petri dish). The
germination speed index (GSI) was calculated using the formula proposed by Maguire [
42
]:
GSI = G1/N1 + G2/N2 + . . . + Gn/Nn
in which: G1, G2 and Gn = number of normal seedlings, computed during the first, second
and last counts; and N1, N2, Nn = number of sowing days during the first, second and
last counts.
2.3. Micropropagation
2.3.1. Establishment of Initial Cultures
Ten days after the completion of seed germination, young seedlings (plantlets) were
transferred to Hf medium (8 g L
−1
agar) and to MS medium supplemented with 6-
benzyladenine (BA) (0.5 or 1.0 mg L
−1
) for
in vitro
cultures. Forty (40) days later, the
plantlets were sub-cultured on solid medium supplemented with BA, kinetin (KIN), and
Horticulturae 2022,8, 390 4 of 14
6-
γ
-
γ
-(dimethylallylamino)-purine (2IP) at 0.5 and 1.0 mg L
−1
, or Hf medium. The root
system of young plantlets was excised before culturing on a new MS medium in both
previous stages.
2.3.2. Effect of Explant Type and Medium Type on Shoot Multiplication
During the multiplication stage, lateral shoots and leaves excised from the base of
plantlet stems were cultured on solid or liquid, Hf MS medium; or supplemented with BA,
KIN and 2IP at 1.0 mg L
−1
. Explants were sub-cultured on the same medium as that on
which each had originated. A total of four subcultures followed.
2.3.3. In-Vitro Culture Conditions and Data Collection
In vitro
cultures of the initial and establishment phases were carried out in 60 mL
glass vessels (three explants per vessel); and covered with plastic wrap (Sanitas; Sarantis
S.A., Athens, Greece).
In vitro
liquid, or solid, cultures of the multiplication phase were
conducted in Magenta GA-7 vessels (77 mm
×
77 mm
×
77 mm, Sigma-Aldrich) (four
explants per vessel). All cultures were maintained at 25
◦
C, with a 16 h photoperiod
at 37.5
µ
mol m
−2
s
−1
provided by cool-white, fluorescent lamps. All solid media were
solidified with 8 g L
−1
agar and the pH of all media was adjusted to 5.7–5.8 before addition
of the agar and autoclaving (121
◦
C for 20 min). Data on the initial cultures were collected
after 40 days of culture. In compliance with previous studies [
30
], data on the establishment
and multiplication phase were collected after 60 days of culture. Data were also collected
on shoot proliferation percentages, shoot numbers per explant, shoot lengths, and number
of leaves per shoot. To obtain the proliferation potential of the cultures, the “multiplication
index” (MI) of each culture was calculated by multiplying the percentage of explants
that produced shoots by the mean number of shoots per responding explant. Rooting
percentages and root numbers and lengths were recorded during the rooting experiments.
2.3.4. Ex Vitro Acclimatisation
Rooting and shooting took place at the same time. Rooted microshoots 2.0–2.5 cm long,
on various media, separated from shoot-clusters and thoroughly rinsed under running
tap water in order to remove the medium before being transferred to 500 mL containers
(eight plantlets per container), on peat (pH 5.5–6.5, Klasmann-Delimann Gmbh, Geeste,
Germany) and perlite (particles diameter 1–5 mm, Perloflor, Isocon S.A., Athens, Greece)
substrate 1:1 (v/v). All containers were covered with transparent plastic wrap (Sanitas) to
maintain humidity. Pots were then placed for one week in a growth chamber (25
◦
C and
16-h cool white fluorescent light 37.5
µ
mol.m
−2
s
−1
/8-h dark photoperiod). Next, pots were
uncovered for one week more. Afterwards, pots were transferred to a heated glasshouse
(37
◦
58
0
58.0
00
N, 23
◦
42
0
19.2
00
E) and placed on a greenhouse bench for another 7 days. At the
end of that period, data on acclimatisation were recorded. The plants were transplanted
singly in 500 mL plastic pots with peat: perlite (1/1, v/v) and fertilized monthly with
2.0 g L−1
complete water-soluble fertilizer (Nutrileaf 60, 20-20-20; Miller Chemical and
Fertilizer Corp., Hanover, PA, USA). The final survival rate was checked two months later.
2.4. Statistical Analysis
Our statistical analysis used the completely randomized design (CRD) method. The
significance of the results was tested by one- or two-way analysis of variance (ANOVA).
Data on percentages were arcsine-transformed prior to statistical analysis. The treatment
means were compared via use of the Student’s t-test at p
≤
0.05 (JMP 14.0 software, SAS
Institute Inc., Cary, NC, USA, 2013). As shown on the data tables, the number of replicates
per treatment differed between experiments. Experiments on the multiplication phase
involved four subcultures. Data for the purposes of our statistical analysis were pooled.
Horticulturae 2022,8, 390 5 of 14
3. Results
3.1. Seed Germination
The disinfection method of seeds resulted in 100% of success. The germination per-
centages were high at 15, 20, and 25
◦
C, i.e., 86.00, 89.00, and 84.00%, respectively (Table 1;
Figures 2and 3), with no statistical differences. Germination at 25
◦
C was completed in
3 days. At 30
◦
C the germination percentage was 35.00%. Cardinal germination temper-
atures were at 15 and 30
◦
C (86 and 35% germination, respectively). T
50
was completed
in 2 days at 25
◦
C. Seeds for that treatment had a higher germination speed index (191.51)
(Table 1).
Table 1. In vitro
germination of Dyckia brevifolia seeds, T
50
and germination speed index (GSI) at
temperatures shown, after six months of storage at room temperature.
Temperature (◦C) Germination (%) ±SD * T50 (Days) GSI
5 0 - -
10 0 - -
15 86.00 ±8.21 a 15 25.24 c
20 89.00 ±5.47 a 5 135.33 ab
25 84.00 ±11.81 a 2 191.51 a
30 35.00 ±16.62 b 4 84.17 b
35 0 - -
*
Different letters in the same column indicate significant differences. Mean (
±
SE) separation in columns by
Student’s t-test at p≤0.05, n= 5, 20 seeds/Petri dish (total 100 seeds per treatment).
Figure 2.
Germinated seeds on MS, Hf, of Dyckia brevifolia at 25
◦
C after 3 (
A
) and 20 (
B
) days of
culture in Petri dishes. Bars represent 1.0 cm of length.
Figure 3.
Germination time course curves of Dyckia brevifolia seeds as affected by temperature. Five
replicates of 20 seeds per treatment were used.
Horticulturae 2022,8, 390 6 of 14
3.2. Micropropagation
3.2.1. Initial Culture and Establishment
D. brevifolia plantlets deriving from
in vitro
grown seedlings were transferred on MS
medium supplemented with BA at 0.5 or 1.0 mg L
−1
or Hf medium to continue their
growth (initial culture). There was no difference between the treatments, and the 40-day
plantlets were 0.7 cm in height, and showed vigorous growth, of five to six leaves. In the
subsequent establishment stage, the survival of plantlets was 95–100%. The plantlets were
more vigorous, with more leaves than previously, i.e., during the initial phase (Figure 4A).
They were similar in height (0.6–0.8 cm) in most of the media tested, with the exception of
the medium containing 0.5 mg L
−1
BA on which the growth of shoots was the shortest one
(0.5 cm). The number of leaves was higher (8.4 leaves) on a Hf medium and presented no
difference from the MS medium supplemented with KIN or 2IP at 1.0 mg L−1(Table 2).
Figure 4. In vitro
regeneration of Dyckia brevifolia: (
A
) plantlets growing and root formation during
initial transfer of young seedlings after forty days on solid MS-Hf medium; (
B
) first shoots forming on
shoot explants after two weeks of culture on solid MS medium containing BA 1 mg L
−1
; (
C
) multiple
shoot formation on a shoot explant after six weeks on a solid MS medium with 1 mg L
−1
BA;
(
D
) multiple shoot and spontaneous root formation on a shoot explant after six weeks of culture on
a liquid MS medium with 1 mg L
−1
2IP; (
E
) first shoots forming on leaf- explants after two weeks
of culture on a solid MS medium containing BA 1 mg L
−1
; (
F
) multiple shoot and spontaneous root
formation on a shoot explant after six weeks of culture on a solid MS medium with 1 mg L
−1
2IP;
(
G
) plantlets during ex vitro acclimatisation; (
H
) two-month-old plants showing vigorous growth.
Bars represent a length of 1 cm.
Horticulturae 2022,8, 390 7 of 14
Table 2.
Effect of cytokinin type and concentration on shoot proliferation from shoot explants excised
from shoots produced on MS medium (Hf or containing BA at 0.5 or 1.0 mg).
Cytokinin Concentration
(mg L−1)
Survival
(%)
Growth
(cm)
Number of
Leaves
Hf †- 95.0 a 0.7 ab 8.4 a
BA 0.5 100.0 a 0.5 c 6.5 c
1.0 97.5 a 0.6 abc 6.4 c
KIN 0.5 97.5 a 0.8 a 6.8 bc
1.0 95.0 a 0.6 abc 7.3 ab
2IP 0.5 100.0 a 0.7 abc 6.1 c
1.0 100.0 a 0.7 abc 7.6 ab
Fone-way ANOVA NS *** ***
Fcyt NS
Fconc NS
Fcyt×conc NS * *
Different letters in the same column indicate significant differences. NS, *, *** Nonsignificant or significant at
p≤0.05, p≤0.001, respectively, n= 40–50. †Hf (hormone free) treatment was excluded for 2-way ANOVA.
3.2.2. Multiplication Stage
Shoot Explants
The multiplication stage comprised a total of four subcultures so as to ascertain
whether growth would be optimized in: (a) solid MS containing BA, KIN, or 2IP, at
1.0 mg L−1
, or Hf; (b) liquid MS containing BA, KIN, or 2IP, at 1.0 mg L
−1
, or Hf. The
two-way analysis showed that the liquid media were superior to the solid ones in terms
of growth (height of plantlets), leaf number/main shoot, number of lateral shoots and
MI (Table 3). The plantlets showed sturdy growth and were vigorous with lateral shoots
(Figure 4B,C,D). The type of cytokinin used also played a significant, positive role. The
maximum MI (1.93 and 1.97) was recorded on liquid media supplemented with 2IP or BA
at 1.0 mg L
−1
(Table 3, Figure 4B). The percentage of lateral shoot formation was higher on
solid and liquid media containing BA (83 and 73%, respectively) and on liquid media with
2IP (84%) (Figure 4D). The higher lateral shoot number (2.7) was observed in the Hf liquid
medium with BA (Table 3, Figure 4C). More leaves were formed on Hf MS, and the higher
growth was estimated when explants were cultured on MS containing KIN or 2IP (2.3 and
2.2 cm, respectively).
Table 3.
Effect of cytokinin type and concentration on shoot proliferation from shoot explants, excised
from plantlets on solid and liquid media containing BA, KIN, or 2IP, at 1.0 mg L−1, or Hf.
MS Cytokinin
Stem
Growth
(cm)
Number of
Leaves/Main
Shoot
Formation of
Lateral Shoots
(%)
Lateral
Shoot
Number
MI
Number of
Leaves/Lateral
Shoot
Solid Hf †0.9 b 8.5 cd 0.0 c 0.0 c - 0.0 c
BA 0.8 b 6.2 e 83.0 a 1.8 ab 1.50 ab 4.6 b
KIN 0.9 b 7.7 de 0.0 c 0.0 c - 0.0 c
2IP 0.7 b 6.1 e 54.0 b 2.0 ab 1.08 b 4.9 b
Liquid Hf †1.7 ab 12.1 a 8.0 b 1.5 bc 0.11 b 4.9 b
BA 1.9 ab 10.2 bc 73.0 a 2.7 a 1.97 a 4.9 b
KIN 2.3 a 11.6 ab 63.0 ab 1.9 ab 1.19 b 6.1 a
2IP 2.2 a 10.5 bc 84.0 a 2.3 ab 1.93 a 6.0 a
Fone-way ANOVA *** *** ** *** * ***
Fmed *** *** *** ** ***
Fcyt NS ** *** ** ***
Fcyt×med NS NS * NS NS NS
Different letters in the same column indicate significant differences. NS, *, **, *** Non-significant or significant at
p≤0.05, p≤0.01, p≤0.001, respectively, n= 50–60, †Hf = hormone-free.
Horticulturae 2022,8, 390 8 of 14
Leaf Explants
Bud proliferation started at the base of both shoot and leaf explants 2 weeks after
culture initiation (Figure 4B,E). Regarding leaf explants, shoots originated directly from
protuberances which had formed at the leaf blade’s cut without any intermediate callus
phase. Two-way analysis revealed that solid media were more effective than liquid media,
as lateral shoot formation was higher (Table 4). On the other hand, explants cultured in
liquid media, gave rise to plantlets of greater growth. Multiple shoots formed in all media,
but the lateral shoot formation was higher on solid MS supplemented with BA, KIN or
2IP (41.0, 35.0 and 44.5%, respectively). Lateral shoot number stood at 7.4 and the MI was
higher on liquid media containing BA, registering 1.9 (Table 4).
Table 4.
Shoot proliferation from leaf-origin explants excised from shoots produced either on Hf MS
medium or on MS containing BA at 0.5 or 1.0 mg L
−1
, on solid and liquid media containing BA, KIN
or 2IP at 1.0 mg L−1or Hf.
MS Cytokinin
Formation
of Lateral
Shoots (%)
Growth
(cm)
Lateral Shoot
Number MI Number of
Leaves/Shoot
Solid Hf †20.0 b 1.0 ab 1.4 c 0.28 c 8.8 a
BA 41.0 a 0.5 c 1.4 c 0.57 c 7.0 a
KIN 35.0 a 0.8 bc 1.5 c 0.53 c 8.0 a
2IP 44.5 a 0.9 b 1.5 c 0.67 bc 8.9 a
Liquid
Hf†17.0 b 1.2 ab 1.0 c 0.17 c 7.4 a
BA 26.0 ab 1.0 ab 7.4 a 1.90 a 6.6 a
KIN 22.5 b 1.3 a 2.6 b 0.59 bc 7.6 a
2IP 27.5 b 1.0 ab 3.3 b 0.90 b 6.3 a
Fone-way ANOVA *** ** *** ** NS
Fmed *** *
Fcyt NS NS
Fcyt×med NS NS * * *
Different letters in the same column indicate significant differences. NS, *, **, *** Non-significant or significant at
p≤0.05, p≤0.01, p≤0.001, respectively, n= 50–60. †Hf = hormone-free.
3.2.3. In Vitro Rooting and Ex Vitro Acclimatisation
Shoot-origin plantlets produced on establishment media, rooted spontaneously at a
high percentage (95%) on Hf medium, or supplemented with 0.5–1.0 mg L
−1
KIN or 2IP
(Table 5). Rooted plantlets produced 2.1 and 2.3 roots per plantlet of 1.2 and 1.1 cm length
on Hf and MS containing 0.5 mg L
−1
KIN, respectively. During the multiplication phase,
rooting was continuous. Shoot- and leaf-origin plantlets rooted at a higher percentage (94.0
and 74.5%, respectively) on a solid MS medium (Table 6, Figure 4F). The percentage of
rooting was high on liquid HF or MS medium supplemented with 1 mg L
−1
2IP (74.5 and
77.5%, respectively) for shoot-origin shoots. Leaf-origin shoots rooted at a lower percentage
(Table 6). Rooted microshoots were acclimatised ex vitro at 100% (Figure 4G). Acclimatised
plants were transplanted in pots. Their survival rate while growing was 100% after two
months (Figure 4H). Lateral sprouts were formed at the base of some plantlets during the
acclimatisation phase (Figure 5).
Horticulturae 2022,8, 390 9 of 14
Table 5. In vitro
rooting of shoots during first subculture on MS supplemented BA, KIN or 2IP at 0.5
or 1.0 mg L−1, or Hf.
Cytokinin Concentration
(mg L−1)Rooting (%) Root Number Root Length (cm)
Hf †- 95.0 a 2.1 a 1.2 a
BA 0.5 20.0 b 1.0 b 0.5 c
1.0 13.0 b 1.0 b 0.5 c
KIN 0.5 93.0 a 2.3 a 1.1 ab
1.0 81.0 a 1.6 ab 0.9 bc
2IP 0.5 100.0 a 1.3 b 0.9 bc
1.0 79.0 ab 1.6 ab 0.8 c
Fone-way ANOVA ** ** **
Fcyt NS
Fconc **
Fcyt×conc * * NS
Different letters in the same column indicate significant differences,
†
Hf = hormone-free. NS, *, ** Non-significant
or significant at p≤0.05, p≤0.01, respectively, n= 50–60.
Table 6. In vitro rooting of shoots derived from shoots or leaves as affected by the type of MS (solid
or liquid) and of cytokinin during multiplication stage.
Shoot-Origin Leaf-Origin
MS Cytokinin Rooting
(%)
Root
Number
Root Length
(cm)
Rooting
(%)
Root
Number
Root Length
(cm)
Solid Hf †94.0 a 2.2 b 1.1 c 73.5 a 2.0 a 1.2 a
BA 0.0 c 0.0 c 0.0 c 50.0 ab 1.0 a 0.5 a
KIN 47.0 b 1.4 b 1.0 c 18.0 b 1.7 a 1.0 a
2IP 43.0 b 1.8 b 0.9 c 52.5 ab 2.0 a 0.9 a
Liquid Hf †74.5 ab 4.2 a 1.7 b 62.5 ab 2.4 a 1.4 a
BA 0.0 c 0.0 c 0.0 c 0.0 b 0.0 b 0.0 b
KIN 18.3 b 3.9 a 2.3 a 0.0 b 0.0 b 0.0 b
2IP 77.5 ab 3.8 a 2.4 a 0.0 b 0.0 b 0.0 b
Fone-way ANOVA *** *** *** * NS NS
Fmed ***
Fcyt NS
Fcyt ×med *** NS * ** *‘ *
Different letters in the same column indicate significant differences. NS, *, **, *** Non-significant or significant at
p≤0.05, p≤0.01, p≤0.001, respectively, n= 50–60. †Hf = hormone-free.
Figure 5.
Lateral sprouts formed at the base of one of the plantlets during acclimatisation. Bar
represents length of 1 cm.
Horticulturae 2022,8, 390 10 of 14
4. Discussion
The small size of Bromeliaceae seeds and their diminished reproduction capacity [
43
,
44
]
has resulted in scientists’ turning to alternative propagation by means of tissue culture-
based techniques, starting with young tissues of
in vitro
grown seedlings. With regard to
the temperature range for seed germination of the D. brevifolia, the present study is the first
one to investigate and define cardinal temperatures as ranging from 15
◦
C to 30
◦
C. At
temperatures of 20–25
◦
C, D. brevifolia exhibited maximum germination in a brief period
of time. The present study’s finding has been confirmed by Paula et al. [
36
] who recently
evaluated the effect of liquid nitrogen during cryopreservation of D. brevifolia seeds and
determined a germination percentage of 95% at 25
◦
C. At the same time, Moresco et al. [
37
]
have also cited a high germination rate. Such references lead to the thought that fast
germination of D. brevifolia may prove an adaptive strategy of taking advantage of the short
period of rainfall and water availability that D. brevifolia has at its disposal in its natural
habitat. That strategy has been reported as successful for Haloxylon recurvum Bunge ex.
Boiss, for H. salicornicum Bunge ex. Boiss [
45
], and for Limonium axillare (Forssk.) Boiss [
46
].
The study also indicates that D. brevifolia seeds have no dormancy period as reported
for D. distachya, another Dyckia species [
28
]. With regard to germination percentages
of two other Dyckia species, it has been observed that D. distachya seeds germinate at
higher percentages when the temperature range is 20–30
◦
C [
47
], in the presence of light.
Similarly, seeds of Dyckia tuberosa have shown a higher germination percentage at a range of
30–35 ◦C [48]
, with cardinal temperatures for germination ranging from 10
◦
C to 40
◦
C. The
lower optimum temperature for germination of D. brevifolia may be explained by the major
impact of the environment on seed germinability during seed production [49]. Regarding
the optimum temperatures for germination of Mediterranean species overall, the usual
range is between 15–20
◦
C [
50
–
52
]. The range of germination of D. brevifolia, which is similar
to that of other Mediterranean species, could enhance its use as an ornamental plant, as is
the case with D. encholirioides, another endangered species, which has no limitations when
germinated
in vitro
, in contrast to its germination in a natural environment where there
are certain obstacles that could reduce its germination rate [
53
,
54
]. It could also lead to the
introduction of new varieties/types into the Mediterranean Basin countries: plants grown
from seeds are characterised by high quality that could lead to the production of disease-
and/or virus-free plants enjoying a prolonged lifespan [
55
].
In vitro
propagated plants
should be indexed as being free of viruses and virus-like diseases through enzyme-linked
immunosorbent assay (ELISA) and molecular methods [
56
]. Moreover, seed propagation is
the basis for production of commercially attractive agronomic and horticultural plants [
57
].
Thus, using seeds as the starting material could meet the increasing demand for ornamental
succulent plants [58].
Many areas around the planet face strong anthropogenic pressure [
59
]. Inevitably as
well as regrettably, several species will be included in the official lists of threatened species.
It is quite the paradox that, although certain species are faced with extinction in their natural
habitat, those very species are widely used in ornamental horticulture and by landscape
designers. Cases in point, Origanum dictamnus, a plant endemic to Crete, Greece, [
60
]
or Neoregelia cruenta, an endemic bromeliad found exclusively on the sandy parts of the
coastal plain of Rio de Janeiro [
22
,
61
].
In vitro
techniques are of paramount importance
for conservation purposes. In view of that, an efficient micropropagation protocol for D.
brevifolia could enhance as well as accelerate efforts to that direction. In the present study,
initial culture and establishment phases were successful for explants derived from young
seedlings, providing an effective method of production of
in vitro
-grown plantlets. An
effective plant tissue culture protocol is based on the selection of the appropriate explant
type [
62
]. The present study has shown that shoot-origin explants can lead to a successful,
direct-shoot regeneration on solid or liquid media supplemented with BA at 1.0 mg L
−1
.
Of particular interest is the ability of the explants to produce lateral shoots with a high rate
of spontaneous rooting on liquid media with 2IP (1.0 mg L
−1
) at the same time. Regarding
leaf origin-explants, shoots originating directly from protuberances located at the cut end
Horticulturae 2022,8, 390 11 of 14
of the leaf blade, without any intermediate callus phase, produced multiple shoots both in
liquid and solid media. Bud and protuberance formation at the base of leaf explants prior
to bud development has been described in the case of Dyckia macedoi [9].
The relevant literature indicates that successful propagation systems of other bromeli-
ads do exist. Those systems are based on tissue culture through liquid media on which the
number of leaf-derived buds could reach double the number of buds as produced on solid
media with the same composition [
63
]. Similar base leaf regeneration in bromeliads was
also observed on the base of leaf sheaths removed from greenhouse-grown seedlings of
Puya tuberosa [
64
]. In the present study, the shoot production that took place by leaf-derived
explants on a liquid MS medium supplemented with 1.0 mg L
−1
BA was exceptionally
high (7.4 shoots/explant). On the other hand, the percentage of lateral shoot formation
was a mere 26.0%, since the task of excising a specific leaf explant that could be used as a
suitable explant successfully proved particularly daunting.
The ultimate survival of acclimatised plantlets is crucial for a successful
in vitro
propagation protocol. In the present study, the percentage of survival after two months
reached a full 100%, an acclimatisation rate which is quite high and observed also in the
case of D. distachya [
27
], and of D. agudensis [
29
]. The present experimentation resulted in an
efficient plant propagation method which could be used either for commercial propagation
of selected clones or for in vitro and ex situ conservation programs.
5. Conclusions
In conclusion, the present study investigated a fully reliable procedure for propagation
on D. brevifolia starting from a small quantity of plant material. In quite a short period,
three-month-old seeds of D. brevifolia germinated profusely, at 15 to 25
◦
C. Cardinal tem-
peratures for germination were defined at 15
◦
C and 30
◦
C. Regarding micropropagation,
seedling-origin shoot explants responded more eagerly than single leaves to
in vitro
culture.
Nevertheless, both exhibited high shoot multiplication on liquid MS medium supplemented
with 1.0 mg L
−1
2IP, with simultaneous rooting. Microshoots rooted abundantly on Hf,
half-strength MS medium and were successfully established at ex vitro conditions.
In many countries, ornamental bromeliads fetch a high market price as they are sought
after by the fields of floriculture and landscape architecture. To our knowledge, this is the
first report on
in vitro
propagation of D. brevifolia. This experimental procedure leads to
the production of a high number of individuals, independently of the natural vegetative
cycle of D. brevifolia in the wild. This type of propagation may facilitate both the needs for
increased demand that floriculture or ornamental horticulture face and in producing new
specimens for
in vitro
and ex situ conservation purposes. Starting with young tissue taken
from
in vitro
germinated seeds is essential for the preservation of the genetic diversity that
threatened species are in need of. Future studies on genetic stability could evaluate the use
of regenerated plants for reintroduction in those plants’ natural habitats.
Author Contributions:
Conceptualization, K.B.; methodology, K.B. validation, K.B.; formal analysis,
K.B.; investigation, K.B.; resources, K.B. and K.-P.P.; data curation, K.B. and K.-P.P.; writing—original
draft preparation, K.B.; writing—review and editing, K.B.; visualization, K.B.; supervision, K.B.;
project administration, K.B.; funding acquisition, K.B. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments:
The authors would like to thank the plant nursery Gryllis Water Lilies (POBOX
Z2-04, Vranas—Marathon Attica, Greece) for donating the seeds of the species investigated.
Conflicts of Interest: The authors declare no conflict of interest.
Horticulturae 2022,8, 390 12 of 14
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