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In vitro multiplication and genetic stability of two species of Micranthocereus

Backeb. (Cactaceae) endemic to Bahia, Brazil

Article  in  Plant Cell Tissue and Organ Culture · December 2017

DOI: 10.1007/s11240-017-1304-6

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Plant Cell Tiss Organ Cult
DOI 10.1007/s11240-017-1304-6
ORIGINAL ARTICLE
In vitro multiplication and genetic stability of two species
of Micranthocereus Backeb. (Cactaceae) endemic to Bahia, Brazil
L. M. Civatti1   · M. N. G. Marchi3 · A. S. Schnadelbach1,2 · M. C. Bellintani1
Received: 6 January 2017 / Accepted: 31 August 2017
© Springer Science+Business Media B.V. 2017
Abstract  The two targeted species of this study, Micran-
thocereus flaviflorus subsp. densiflorus and M. polyan-
thus subsp. alvinii, are endemic to the state of Bahia and
have ornamental value. The main goal of this work was to
micropropagate these species and to evaluate the genetic
stability of the regenerated plants. To do so, shoots origi-
nated from in vitro germinated plants were inoculated in
MS/2 (Murashige, Skoog, Physiol Plant 15:473–497, 1962)
media containing 1.34 μmol L−1 of α-naphthaleneacetic acid
(NAA) for morphogenesis induction. This was repeated for
three consecutive subcultures, and the subcultured shoots
were designated by order of production as S1, S2 and S3.
Retention of morphogenic potential and acceleration of orga-
nogenic response was observed after the three subsequent
propagation events, so that if shoots were used as explant
source the in vitro propagation of M. flaviflorus could be
Communicated by T. Winkelmann.
Electronic supplementary material  The online version of this
article (doi:10.1007/s11240-017-1304-6) contains supplementary
material, which is available to authorized users.
* L. M. Civatti
laila.civatti@gmail.com
1
Programa de Pós Graduação em Genética e Biodiversidade,
Universidade Federal da Bahia, Rua Barão de Geremoabo,
147 ‑ Campus de Ondina, Salvador, Bahia CEP 40170‑290,
Brazil
2
Programa de Pós Graduação em Recursos Genéticos
Vegetais, Universidade Estadual de Feira de Santana,
Avenida Transnordestina S/N ‑ Novo Horizonte,
Feira de Santana, Bahia CEP 44036‑900, Brazil
3 densiflorus (Buining and Brederoo) P.J. Braun and Este-
Instituto Federal de Educação, Ciência e Tecnologia
Baiano, Rua Barão de Camaçari, 118 ‑ Centro, Catu,
Bahia CEP 48110‑000, Brazil
 
achieved in 90 days and that of M. polyanthus could be opti-
mized to 60 days of duration. In order to perform genetic
stability analysis along subcultures, ISSR markers were used
and genetic variation between shoots of each subculture and
their donor plant was measured with Jaccard’s similarity
coefficient. This analysis revealed high genetic stability in
the in vitro propagation of all the donor plants of M. fla-
viflorus and M. polyanthus in regards to three consecutive
shoot subcultures, in which similarities were 100% for both
species. The study of a greater number of subcultures is sug-
gested to assess morphogenesis potential and genetic fidelity
in long term.
Keywords  Cactaceae · ISSR · Molecular markers ·
Micropropagation · Plant growth regulators · Somaclonal
variation
Introduction
The adaptations of plants belonging to family Cactaceae,
such as the morphology of their cladodes and spines, as
well as their flowers, are motives for their collection and
cultivation worldwide (Goettsch et al. 2015). In addition,
cactus populations are threatened by the devastation of their
habitats as well (Taylor and Zappi 2004). Therefore, propa-
gation and conservation studies in Cactaceae are a necessity
(Goettsch et al. 2015).
According to Charles (2009), the eight species of Micran-
thocereus possess ornamental potential and are, therefore,
subject to commercialization and threats created by illegal
collection to that end. Micranthocereus flaviflorus subsp.
ves, is a cactus endemic to the state of Bahia, Brazil, with a
restricted distribution (Taylor and Zappi 2004). This species
13
Vol.:(0123456789)

is listed in the Appendix II of CITES (Convention on Inter-
national Trade in Endangered Species of Wild Fauna and
Flora) and is classified by the IUCN (International Union for
Conservation of Nature) Red List of Threatened Species as
“near threatened” (CITES 2016; Machado and Braun 2013).
The other species of the same genus targeted in this work,
M. polyanthus subsp. alvinii M. Machado and Hofacker, also
has a restricted distribution being endemic to Bahia, except
it is rare and classified as “threatened” by the IUCN Red
List of Threatened Species and the Brazilian Ministry of
the Environment (MMA 2008; Machado et al. 2013). Since
in situ conservation for these species is lacking, M. flaviflo-
rus and M. polyanthus were objects of ex situ conservation
efforts, including studies of in vitro establishment through
seeds collected at natural populations, along with storage
and cryopreservation of such seeds and micropropagation
of in vitro germinated plants (Veiga-Barbosa et al. 2010;
Civatti et al. 2015a, b, 2017).
In vitro multiplication using plants germinated in lab-
oratory can supply the demand of the ornamental market
while protecting the integrity of natural populations indi-
rectly, and still provide plants for reintroduction initiatives.
Genetic fidelity studies have demonstrated, however, that
micropropagation can generate genetically distinct plants, a
phenomenon known as somaclonal variation (Khattab et al.
2014). Works with Inter Simple Sequence Repeat (ISSR)
markers have been efficient in detecting somaclonal vari-
ation in numerous cacti, for example, Hylocereus undatus
(Haw.) Britton & Rose (Fan et  al. 2013), Pilosocereus
robinii (Lem.) Byles & G.D. Rowley (Khattab et al. 2014),
Melocactus glaucescens Buining & Brederoo (Torres-Silva
2014), and Stephanocereus luetzelburgii (Vaupel) N.P. Tay-
lor & Eggli (Marchi 2016).
Considering that in literature there are no studies of long
term conventional or in vitro propagation of M. flaviflorus
and M. polyanthus, the aim of this work was to evaluate
morphogenesis along three successive in vitro multiplica-
tion events and determine if somaclonal variation is found
within three subcultures of shoots from the targeted species.
Materials and methods
Initial plant material
Seeds of Micranthocereus flaviflorus subsp. densiflorus and
M. polyanthus subsp. alvinii obtained from mature fruits
were disinfested in laminar flow cabinet by immersing
seeds in absolute alcohol for 1 min, followed by soaking in
sodium hypochlorite at 2.5% for 15 min, and then rinsing
three times with sterile water before sowing in half-strength
Murashige and Skoog (1962) medium (MS/2) at 25 ± 3 °C.
Plants obtained in this manner were maintained in MS/2
13
Plant Cell Tiss Organ Cult
media until the setting up of experiments, with periodical
transfers to fresh media to allow growth and sustainment.
These in vitro germinated plants of M. flaviflorus and M.
polyanthus, 18 and 15 months old respectively, were used
as explant source for in vitro multiplication.
Culture conditions
Culture conditions were established in growth room with a
16-h photoperiod under an irradiance of 60 μmol m−2 s−1
supplied by fluorescent lamps of cold white light (Duramax,
40W) at a constant temperature of 25 ± 3 °C.
In vitro multiplication of M. flaviflorus and M.
polyanthus
The nutrient medium used for in  vitro multiplication
was MS/2 supplemented with 15  g  L−1 of sucrose and
1.34 μmol L−1 of α-naphthaleneacetic acid (NAA), gelified
with 6 g L−1 of Agar and chemically sterilized with sodium
hypochlorite containing 2.5% of active chloride (adapted
from Teixeira et al. 2006) (Civatti et al. 2017). The in vitro
germinated plants were cut crosswise, apexes and roots were
desconsidered, and explants were then placed on the nutrient
medium aforementioned. Shoots obtained at each multiplica-
tion event were detached from their explants and inoculated
in MS/2 free of plant growth regulators under the same cul-
ture conditions. After two to three transfers in said medium,
these shoots were themselves cut crosswise, desconsidering
apexes and roots, and placed in medium supplemented with
1.34 μmol L−1 of NAA for morphogenesis induction. This
was repeated for three consecutive subcultures, and subcul-
tures shoots were named in chronological order S1, S2 and
S3. Threfore, in vitro germinated plants that were multi-
plied using NAA at 1.34 μmol L−1 (called donor plants, or
D) originated S1 shoots, which then went through the same
process to originate the S2 shoots, and these similarly origi-
nated the S3 shoots.
The retainment of morphogenic potential was evaluated
along three consecutive cycles of in vitro multiplication.
Means of the variables number of shoots per explant, per-
centage of shooting explants, percentage of rooting explants,
and percentage of surviving explants were calculated at
each in vitro multiplication event, in which parcial evalua-
tions were made every 30 days achieving 120 days for each
event. In these multiplication events ten replicates of five
explants were used as donor plants (D) and ten replicates of
six explants were used for the following subcultures (S1, S2
and S3), for each species.
For the temporal analysis of in  vitro morphogenesis,
the variables number of shoots per explant and shooting
explants (%) were measured at every partial evaluation
of in vitro multiplication using the same sample design
Plant Cell Tiss Organ Cult
aforementioned. The means of these variables for each eval-
uation (30, 60, 90 and 120 days) were compared in every
in vitro multiplication event (namely M1, M2 and M3, in
chronological order).
The number of shoots produced per explant was com-
pared considering all donor plants of both species, a variable
consisting of the arithmetic mean of shoots generated by
all explants of each donor plant during every multiplication
event. For each donor plant, three replicates of three explants
each had their shoot production accounted for.
Genetic stability analysis
To assess genetic stability in M. flaviflorus and M. polyan-
thus each donor plant and shoots originated from it for three
subsequent subcultures were analysed. For every donor plant
(D) one shoot of each subculture was analysed, compairing
the D plant to its shoots of each subculture in electrophoresis
gel. In total, 19 donor plants (D) and one shoot originated
from each subculture (S1, S2 and S3) for every one of them
were evaluated for M. flaviflorus (76 plants in all), and so
were 18 donor plants and their shoots from M. polyanthus
(72 plants altogether).
DNA extraction
Regenerated apexes of plants multiplied at every subculture
were sources of plant material used for DNA extraction.
Genetic material was extracted from the samples using small
amounts of plant tissue (5 mg) through Doyle and Doyle’s
(1987) protocol adapted to microtubes. The extracted
DNA was purified utilizing an ammonium acetate protocol
(Patrocínio, E., personal communication). To perform this
purification, 50 μL of DNA were diluted in 550 μL of pH
8.0 Tris–EDTA (TE buffer) at room temperature for 1 h.
Afterwards, 200 μL of ammonium acetate at 7.5 M were
added and the microtubes were homogenized and incubated
in ice for 15 min. Next, the microtubes were centrifuged for
10 min at 8.000 RPM and the supernatant was transferred
to a new microtube, to which 800 μL of cold absolute etha-
nol were added, and the solution was homogenized. These
microtubes were then incubated for one hour at −20 °C and
subsequently centrifuged for 10 min at 12.000 RPM. After
two washes with 70% ethanol the DNA pellets were dried at
room temperature overnight. These pellets were resuspended
in 50 μL of TE for 48 h at room temperature.
Following extraction, DNA analysis was performed
through spectrophotometer quantification (L-QUANT, Loc-
cus Biotecnologia) and through electrophoresis in 1% aga-
rose gel in 1× Tris–EDTA (TAE) buffer. The band pattern
formed was observed staining the DNA with GelRed in the
presence of ultraviolet light.
DNA amplification
For DNA amplification, 20 ISSR primers described by Wolfe
et al. (1998) were tested and the JOHN, MAO, MANNY,
814, 844 and 899 primers were selected. Amplification reac-
tions were executed in a total volume of 20 μL containing
1× reaction buffer (500 mM KCl, 100 mM pH 8.4 Tris–HCl,
1% Triton X-100), 2.5 mM of ­MgCl2, 12 pmol of primer,
200 µM of dNTPs, 1 U of Taq polymerase, ultrapure auto-
claved water and DNA (Supplementary material). In the
amplifications with the 814 primer 3.3 mM of ­MgCl2 were
used instead. A control sample without DNA was included
in each amplification to verify the absence of contamination
in the reagents.
The amplification program initialized at 94 °C for 90 s,
followed by 35 cycles at 94 °C for 40 s, 45–51 °C for 45 s
(see Supplementary material), and 72 °C during 1 min and
30 s, proceeded by one cycle at 94 °C for 45 s, 44 °C for
45 s, and a final extention at 72 °C for 7 min. Later on, sam-
ples were submitted to electrophoresis in 2.0% agarose gel,
stained with GelRed and photographed with Wise Capture
II program (version 1.0.0.0) in an ultraviolet light transil-
luminator. Every gel was repeated at least twice for analysis.
Statistical analysis
In vitro multiplication along subcultures of M. flaviflorus
and M. polyanthus
For data analysis, ANOVA was performed and means were
compared with the Tukey Test at 95%, in the statistics pro-
gram SISVAR 5.3 (Ferreira 2008). All data expressed in %
was arcsine-transformed before analysis.
Comparison of number of shoots per explant
between donor plants
ANOVA was performed for data analysis and means of
the number of shoots per explant were compared using the
Scott–Knot Test at 95% of significance, aiming to eliminate
ambiguity in the comparison of means under analysis, in the
statistics program SISVAR 5.3 (Ferreira 2008).
Analysis of genetic stability
The banding patterns in the gels were manually read and
coded as presence (1) and absence (0) of bands in a binary
matrix. Loci with low resolution and without reproducibil-
ity were excluded from analysis. The percentage of plants
with somaclonal variation originated from each subculture
for both species was calculated considering as variation any
difference in the banding pattern between donor plant and
its respective shoots. Genetic variation among plants was
13
 Plant Cell Tiss Organ Cult

Table 1  Morphogenic response of Micranthocereus flaviflorus and

MF39 MF40
M. polyanthus after three consecutive micropropagation events (M1,

5.5 b
M2 and M3)

MP40
2.6 b
Event Shooting (%) Shoots/explant Rooting (%) Survival (%)

3.5 b
M. flaviflorus

MP39
2.4 b
 M1 93.4 a 6.2 a 95.6 a 97.8 a

Table 2  Mean number of shoots per explant of each donor plant from Micranthocereus flaviflorus and M. polyanthus propagated through three in vitro multiplication events

MF38
 M2 93.6 a 5.8 a 92.0 a 93.6 a

4.7 b
 M3 100 a 6.4 a 100 a 100 a

MP37
5.6 a
M. polyanthus

MF37
10.4 a
 M1 97.7 a 3.5 a 100 a 100 a
 M2 100 a 2.9 a 100 a 100 a

MP36
2.5 b
 M3 100 a 3.9 a 100 a 100 a

MF36
6.3 a
Means followed by the same letter do not differ significantly by the

MP35
5.1 a
Tukey test (p > 0.05)

MF34
7.9 a
estimated through Jaccard’s similarity coefficient (Jaccard

MP34
4.3 b
1912) using the Past 3.11 software (Hammer et al. 2001).

MF33
7.7 a

MP33
3.5 b
Results

MF32
7.5 a

MP32
In vitro multiplication of M. flaviflorus and M.

7.3 a
polyanthus

MF31
5.5 b

Means followed by the same letter in each line do not differ significantly by the Scott-Knot test (p > 0.05)
 Identification code of donor plant (MF, Micranthocereus flaviflorus; MP, Micranthocereus polyanthus)
MP31
None of the analyzed variables showed significant differ-

2.1 b
ences between in vitro multiplication events of the studied
MF30
3.4 b
species, which indicates retention of morphogenic potential

MP30
3.3 b
in both cases (Table 1). The shooting, rooting and surviving
rates remained high (over 90%) for the two species, with
MF29
4.5 b

an overall average of 6.1 shoots formed in explants of M. MP29

2.4 b
flaviflorus and 3.4 in M. polyanthus. Neither species pre-
MF28

sented callogenesis or hyperhydricity along the evaluated

6.3 a

multiplication cycles.
MP28
3.4 b

The analysis of the mean number of shoots per explant

MF27

for each donor plant, taking into consideration all three

6.5 a

multiplication events, revealed that different donor plants

MP27
2.9 b

responded differently to propagation with 1.34 μmol L−1 of

MF26
9.1 a

NAA in both species (Table 2). Ten M. flaviflorus donor

plants showed a greater organogenic potential than the rest
MP26
2.6 b

(MF24, MF25, MF26, MF27, MF28, MF32, MF33, MF34,

MF25
7.0 a

MF36 and MF37), while only three of M. polyanthus donor

MP25

plants (MP32, MP35 and MP37) stood out in in vitro shoot

2.7 b

production (Table 2).
MF24
7.3 a

Shoot formation time in each subculture of the two stud-

MP24

ied species indicates there was acceleration in morpho-

2.9 b

genesis from the M1 multiplication event to the M3 event

MF23
3b

(Fig. 1). In the case of M. flaviflorus, the 120 days period

MP23
3.0 b

was necessary for this species to achieve its maximum num-

M. polyanthusa
Shoots/explant

MF21 MF22

ber of shoots per explant only in the first in vitro multipli-

M. flaviflorusa

5.1 b 5.5 b

cation event (M1), since shoot morphogenesis per explant

attained its maximum value earlier in the subsequent events
MP21
3.0 b

(M2 and M3), 90 days after the setting up of the experiments

a

13
Plant Cell Tiss Organ Cult

M. flaviflorus 120 M. flaviflorus

a a
Number of shoots per explant

% of shooting explants
7 a a a 100 a a a
a b
6
a 80 b
5
4 b b 60 b M1
M1
3 b M2
M2 40
2 M3
M3 20
1
c c c c c c c c
0 0
30 60 90 120 30 60 90 120
Time (days) Time (days)

M. polyanthus M. polyanthus
Number of shoots per explant

5 120
a a a a a a

% of shooting explants
a 100 a
4 a a b
a a a 80
3 ab M1
M1 60 b
b M2
2 M2 40
b M3
1 M3 c b
20
b c b c
0 0
30 60 90 120 30 60 90 120
Time (days) Time (days)

Fig. 1  Mean number of shoots per explant and percentage of shoot- Tukey test (p > 0.05), columns of the same multiplication event do not
ing explants of Micranthocereus flaviflorus and M. polyanthus in each differ from each other if signaled with the same letter
in  vitro multiplication event (M1, M2 and M3). According to the

(Fig. 1). Likewise, the explants of this species on the first
propagation event (M1) took 120 days to reach the highest
percentage of shooting explants, which occurred at 90 days
in the following events (M2 and M3), confirming the accel-
eration of morphogenic response in M. flaviflorus (Fig. 1).
For M. polyanthus, on the other hand, the number of
shoots produced per explant found at 60 days on the last
multiplication event (M3) did not differ significantly from
the shooting at 90 or 120 days of that event, indicating a
decrease in response time of this species’ explants, from
90 days to attain maximum number of shoots per explant in
previous propagations (M1 and M2) to 60 days in event M3
(Fig. 1). The same tendency can be observed in the vari-
able percentage of shooting explants, also demonstrating the
quickening of morphogenic response in this species (Fig. 1).
Genetic stability analysis
The six primers used in sample amplifications produced
64 loci, with an average of 10.7 loci/primer. Fragments
showed variation in size of 200 up to 2500 base pairs (bp).
The analysis detected high levels of Jaccard’s genetic simi-
larity between donor plants and their shoots from different
subcultures, with similarities of 100% in both species. No
off-types were observed in any of the subcultured shoots for
either species (Fig. 2).
Discussion
In vitro regeneration is an intricate phenomenon that suf-
fers influences from genetic and environmental factors (Rani
and Raina 2000; Sriskandarajah et al. 2006). In other words,
there is evidence that different plants have intrinsic factors,
be that information of genetic or epigenetic origins, which
interact with external factors existent in plant tissue cul-
ture conditions (Batista et al. 1996; Gurriaran et al. 1999).
The biochemical pathways that control cytokinin homeo-
stasis, for instance, may be affected by characteristics such
as explant, morphology, genotype, and by the culture con-
ditions themselves (Batista et al. 1996; Auer et al. 1999;
Benson 2000). Therefore, the heterogeneous morphogenic
response presented in Table 2, in which donor plants of the
same species demonstrate distinct multiplication rates, is
characteristic of native species, such as M. flaviflorus and
M. polyanthus, known to maintain greater genetic diversity
when compared to those already selected in breeding pro-
grams (Brammer 2002). Hence, for the micropropagation of

13

Fig. 2  Micropropagation of both Micranthocereus flaviflorus (a, c, e)
and M. polyanthus (b, d, f) with media containing 1.34 μmol L−1 of
NAA (bar 1 cm). Donor plants produced S1 shoots in a and b, while
S1 shoots produced S2 shoots in c and d, and S2 shoots produced S3
shoots in e and f  such findings, explants of Pilosocereus gounellei (F.A.C.
such species, donor plants with higher morphogenic poten-
tial must firstly be selected, so that greater efficiency in plant
production may be achieved.
The acceleration in morphogenesis of M. flaviflorus and
M. polyanthus from the donor plants (M1) to the following
shoot subcultures (M2 and M3) may have been caused by
modifications in endogenous plant hormone concentrations
throughout the in vitro multiplication process. Cytokinins
and auxins are central factors in the developmental control
of shoots and alterations in the endogenous levels of these
plant hormones in explants might contribute to an increase
in their regenerative capabilities (Jiménez 2005). The
13
Plant Cell Tiss Organ Cult
balance in endogenous levels of said hormones, however,
may be altered during in vitro multiplication events and thus
determine organogenic potential depending on the species
(Lema-Rumińska and Kulus 2014). In tissue culture, active
phytohormone concentrations in explants are modulated by
exogenous factors (photoperiod, light intensity, temperature,
plant growth regulators, among others) and by metabolism
(Auer et al. 1999; Gurriaran et al. 1999; Benson 2000).
In summary, modifications originated from interactions
between intrinsic factors, like phytohormone concentrations,
and extrinsic ones, like cycles of propagation using plant
growth regulators (NAA at 1.34 µM, in this case), could
have been responsible for altering cell cycle signaling and
thus quickening the shooting process in M. polyanthus and
M. flaviflorus, without loss of morphogenic potential. Such
dynamics were demonstrated with Schlumbergera russeli-
ana × S. truncata, in which an elevated auxin metabolism
in combination with the activity of cytokinin oxidase/dehy-
drogenase favored shooting after a few subcultures (Sris-
kandarajah et al. 2006). For Rhipsalidopsis gaertneri × R.
rosea, the low activity of enzymes with anti-oxidant action
caused recalcitrance (Sriskandarajah et al. 2006).
Another possible explanation for the acceleration
observed in the present work is that of a temporary rever-
sal to a juvenile state in prolonged in vitro tissue cultures,
which is associated to a high morphogenic capacity in spe-
cies of other plant families (Sriskandarajah et al. 1982). This
could explain the fact that, in the multiplication events for
which shoots from previous events (M2 and M3) served as
explant source, propagation transpired more rapidly than in
the events for which the source of explants were in vitro
germinated plants of M. flaviflorus and M. polyanthus
(M1). Such observation was also made for Prunus species,
in which tissues of different ages formed calli with distinct
morphological competences, wherein calli developed from
seedlings were induced into a juvenile state of regeneration,
something that did not occur with calli originated from adult
trees (Druart 1980). On the other hand, in disagreement with
Weber ex K. Schum.) Byles & G.D. Rowley created from
older plants showed greater regeneration capability than
those cut from younger plants (Marchi 2012).
Generally, micropropagation is more successful when
young tissues are used as explant source because they have
superior organogenic competence (Peres 2002). This might
be occasioned by the presence of a great amount of mer-
istematic and undifferentiated cells in said tissues (Shin
et al. 2000), which can be related to their rapid growth and,
thus, better regenerative response (Bispo et al. 2007). Con-
sequently, it is proposed that to decrease propagation time
in both targeted species the plant material used as explant
source be shoots instead of in vitro germinated plants, so
that, in such case, in vitro multiplication of M. flaviflorus is
Plant Cell Tiss Organ Cult
achieved in 90 days and that of M. polyanthus is performed
in 60 days total.
Although there was no increase in shooting production
from one subculture to the next in the studied species, for
other cacti consecutive cycles of propagation augmented
morphogenic response, as was the case of Pelecyphora asel-
liformis Ehrenb., for which the second multiplication event
increased the number of shoots produced by 20–25% (San-
tos-Diaz et al. 2003), and that of S. luetzelburgii’s plants,
which showed higher shoot production after four in vitro cul-
ture cycles (Marchi 2016). The execution of further propaga-
tion cycles using shoots of M. polyanthus and M. flaviflorus
is suggested to verify if there is an increase in morphogenic
potential in these species with more than three succeeding
in vitro multiplication events.
The occurrence of somaclonal variation, here regarded
as the presence of polymorphic bands between donor plant
and its shoots, is dependent upon not only the methodol-
ogy employed to propagate these plants, but also the in vitro
cultivation time, the propagated species and the genotype
of each plant in question (Rani and Raina 2000; Rout et al.
2006; Sharma et al. 2011; Zoghlami et al. 2012). In Cacta-
ceae, studies of genetic stability are still scarce (El-Kazzaz
et al. 1999; Palomino et al. 1999; Mangolin et al. 2002;
Lema-Rumińska 2011; Zoghlami et  al. 2012; Fan et  al.
2013; Hua et al. 2014; Khattab et al. 2014; Torres-Silva
2014; Lopes et al. 2016; Marchi 2016), and no pattern can
be traced for somaclonal variation frequency in species of
this family.
The use of plant growth regulators in high concentrations
or for a prolonged time, together with indirect organogen-
esis, is related to increase in genetic variation rates in tissue
culture (Malda et al. 1999; Rani and Raina 2000; Pérez-
Molphe-Balch and Dávila-Figueroa 2002; Rout et al. 2006;
Torres-Silva 2014). In accordance with this presupposition,
in callus cultures of Cereus peruvianus Mill. kept with
2,4-dichlorophenoxyacetic acid (2,4-D) and high concen-
trations of kinetin (KIN) a genetic variability of 62% up to
96.5% was detected using Random Amplified Polymorphic
DNA (RAPD) markers (Mangolin et al. 2002), the same
as with plants regenerated from calli of Echinocactus gru-
sonii Hildm., also cultivated with 2,4-D (El-Kazzaz et al.
1999), and for calli from Hilocereus undatus generated
with 2,4-D (Lopes et al. 2016). As for P. robinii, exposure
time was short (only 30 days), but the presence of Thidiazu-
ron (TDZ) in the nutrient medium induced high variation
when compared to other treatments with 6-benzylaminopu-
rine (BAP), KIN, 2-isopentenyladenine (2iP) and control,
although shoots were formed through direct organogenesis
(Khattab et al. 2014). And if, on the one hand, the presence
of plant growth regulators may have induced variation in
micropropagated shoots of M. glaucescens, that was not the
only factor acting on the genetic instability of this species,
since in the absence of regulators 25.9% of shoots showed
variation on the ISSR pattern (Torres-Silva 2014). Regard-
ing M. flavilflorus and M. polyanthus, high genetic stability
was found, though studies surveying a greater number of
subcultures are needed to investigate how many subcultures
can be performed consecutively without the emergence of
somaclonal variation.
By using solely NAA as exogenous regulator in the
micropropagation of M. flaviflorus and M. polyanthus direct
organogenesis and high genetic similarity were promoted,
illustrating that low regulator concentrations and direct
organogenesis may uphold in vitro genetic fidelity for three
subcultures in these species. Low concentrations of plant
growth regulators (6-benzyladenine; 0.44 µM) also main-
tained genetic stability in the direct organogenesis of the
bromeliad Puya berteroniana Mez (Viehmannova et  al.
2016). Notwithstanding, studies with other cacti seem to
indicate that the species itself might be a determining factor
concerning the assurance of genetic stability during multi-
plication, regardless of the propagation technique applied
(Rani and Raina 2000; Rout et al. 2006; Sharma et al. 2011;
Zoghlami et al. 2012). Tissue analysis of Copiapoa tenuis-
sima Ritt. forma monstruosa cultivated in the presence of
2,4-D revealed that this species did not have its ploidy lev-
els altered in its shoots, calli or somatic embryos (Lema-
Rumińska 2011). Furthermore, karyotypic stability was
also confirmed for Mammillaria san-angelensis Sánchez-
Mejorada in plants propagated with auxin and kept 7 years
in vitro (Palomino et al. 1999).
Genetic stability was found in other cacti cultures micro-
propagated through direct organogenesis using plant regula-
tors, similarly to what was identified for the donor plants in
M. flaviflorus and M. polyanthus within three shoot subcul-
tures. In cultures of Hylocereus undatus, also by means of
ISSR markers, polymorphic bands were not found between
donor plants and their respective shoots after 15 in vitro
propagation cycles (Fan et al. 2013), which is in accord-
ance with another study in the genus, seeing that after 11
subcultures of in vitro multiplication no polymorphism was
detected between propagated shoots and their donor plants in
Hylocereus sp. using another dominant marker, Start Codon
Targeted (SCoT) (Hua et al. 2014). Plants of Opuntia ficus-
indica (L.) Mill. cultivated in vitro for 5 years were identi-
fied with similarities of 91% and 100% between donor plant
and its shoots using RAPD markers (Zoghlami et al. 2012),
these similarities as high as those found for M. flaviflorus
and M. polyanthus (100%). In view of this, the protocol here
presented may be executed for in vitro multiplication of M.
flaviflorus and M. polyanthus.
Considering the possibility of somaclonal variation
appearance, studies of genetic stability in plant tissue cul-
ture are necessary even in the development of a micropro-
pagation protocol where there is no callogenesis or apparent
13

abnormalities in the obtained plants, as was the case of M.
polyanthus and M. flaviflorus, given that somaclonal varia-
tion is intrinsic to tissue culture and visual indications of its
presence do not always exist (Khattab et al. 2014). Besides,
in face of the diversity exhibited by family Cactaceae, little
is known about somaclonal variation of its species, justify-
ing the persistence of work in this area (Torres-Silva 2014).
Conclusions
Maintenance of morphogenic potential and acceleration in
organogenic response were found along three subsequent
in vitro multiplication events of M. flaviflorus and M. poly-
anthus, and the high genetic similarity between donor plants
and their shoots within three subcultures indicates great
genetic stability in the in vitro propagation of both species.
The in vitro multiplication of M. flaviflorus may be achieved
in 90 days and that of M. polyanthus in 60 days if shoots are
used as explant source. Lastly, the investigation of genetic
stability for further subcultures is suggested to optimize the
propagation process and guarantee genetic fidelity along
more subcultures.
Acknowledgements  The authors would like to thank the Coorde-
nação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for
providing financial support.
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