Scientia Horticulturae 110 (2006) 204–207
www.elsevier.com/locate/scihorti
Micropropagation of the Brazilian endemic bromeliad Vriesea reitzii
trough nodule clusters culture
Glaise Mara Alves a, Lı́rio Luiz Dal Vesco b, Miguel Pedro Guerra c,*
a
Bolsista CNPq/RHAE, LFDGV/FIT/CCA, UFSC, Brazil
Bolsista FAPESP, Programa PIPE, LFDGV/FIT/CCA, UFSC, Brazil
c
Prof. Titular do Laboratório de Fisiologia do Desenvolvimento e Genética Vegetal,
Departamento de Fitotecnia, Centro de Ciências Agrárias,
Universidade Federal de Santa Catarina, Rod. Ademar Gonzaga, 1346,
C. Postal 476, 88.034-001 Florianópolis, SC, Brazil
b
Received 23 June 2005; received in revised form 27 April 2006; accepted 8 June 2006
Abstract
Vriesea reitzii Leme & Costa is an endemic bromeliad from the Atlantic Forest in South Brazil. The devastation of this biome threatens the
extinction of this species that besides its role in the ecosystem has an ornamental value. Tissue culture techniques are important tools for the mass
propagation of threatened bromeliad species. In the present work we established an in vitro regenerative protocol for the large-scale propagation,
and improvement of this species. Young basal leaves used as explants showed 90.6% induction rate of nodule clusters in MS culture medium
supplemented with 20.0 mM 2,4-D and 1.0 mM Kin. The subculture of these nodule clusters to MS medium with BAP, Kin and 2-iP resulted in a
regeneration rate of 60 shoots/g nodule clusters. Subsequent subculture to MS media supplemented with 2.5 mM 2-iP and 0.5 mM NAA and then to
MS medium free of PGR enhanced the full development of plantlets. Plantlets longer than 3 cm were successfully acclimatized showing a survival
rate of 90%.
# 2006 Elsevier B.V. All rights reserved.
Keywords: Bromeliads; Vriesea reitzii; Tissue culture; Regeneration; Histology; Shoot-buds
1. Introduction
The Brazilian Atlantic Forest biome contains high levels of
genetic diversity. Bromeliads are important components of this
biome and among them species of Vriesea sp. are normally
found in the South and Southwest regions of Brazil (Reitz,
1983).
The Vriesea sp. comprises 257 species distributed in the
American continent. In Santa Catarina State it was described 31
native species of Vriesea some of them endemic (Reitz, 1983;
Alves et al., 2004; Rech Filho et al., 2005). Vriesea reitzii Leme
& Costa is an endemic bromeliad occurring in altitudes ranging
from 750 to 1200 m in the states of Santa Catarina, Paraná and
Abbreviations: BAP, 6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; 2-iP, N6(2-isopentyl)adenine; NAA, naphtaleneacetic acid; Kin,
kinetin; PGR, plant growth regulators
* Corresponding author.
E-mail address: mpguerra@cca.ufsc.br (M.P. Guerra).
0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.scienta.2006.06.014
Rio Grande do Sul. It’s name is a tribute to the ‘father of
bromeliads’ the priest Raulino Reitz (Leme and Costa, 1991).
Tissue culture techniques comprise valuable tools for the
mass propagation of bromeliads threatened with extinction
(Pompelli and Guerra, 2004). In this case seeds are used as
explants since they are representative of the genetic structure of
the target population to be conserved. Additionally the in vitro
germination of seeds allows the yield of a large number of
aseptic explants to be inoculated in tissue culture (Mercier and
Kerbauy, 1997).
The basal region of leaves of monocot species as is the case
of bromeliads shows vascular elements that may contain
competent cells for re-differentiation when activated by
regulatory signals, i.e. plant growth regulators (Hosoki and
Asahira, 1980). Such explant types were successfully used in in
vitro systems of several bromeliads (Mercier and Kerbauy,
1997; Carneiro et al., 1999; Alves and Guerra, 2001; Pompelli
and Guerra, 2004).
The present work aimed at the induction and control of in
vitro morphogenesis in V. reitzii in order to establish a
205
G.M. Alves et al. / Scientia Horticulturae 110 (2006) 204–207
regenerative protocol for the large-scale propagation and
improvement.
2. Materials and methods
2.1. Plant material
Seeds of V. reitzii were collected from plants of the
bromeliad collection of the Department of Plant Sciences,
Federal University of Santa Catarina, Santa Catarina State,
South Brazil. The seeds were submitted to disinfestations with
70% ethanol (3 min), sodium hypochlorite 2% (35 min) then
rinsed three times in distilled and autoclaved water. The seeds
were then inoculated in 200 ml glass flasks containing 20 ml of
MS (Murashige and Skoog, 1962) culture medium supplemented with Morel vitamins (Morel and Wetmore, 1951), and
gelled with agar Sigma1 (0.7%, w/v). The pH of the culture
medium was adjusted to 5.8 prior autoclaving at 1.5 atm. at
121 8C during 15 min.
After 8 weeks in culture plantlets 1.5 cm long were transferred
to MS culture medium supplemented with NAA (2.0 mM) and
BAP (4.0 mM) for additional 8 weeks. The cultures were kept in
culture room with temperature of 25 1 8C, 60 5% RH,
37 mE/m2/s light intensity and 16 h photoperiod.
2.2. Induction
Leaf segments (8–10 mm) used as explants were removed
from the basal region of young leaves of in vitro cultured
plantlets with 6 weeks in culture. The explants were inoculated
in Petri dishes containing 25 ml of gelled MS supplemented
with 2,4-D (0, 5, 10 and 20 mM) and Kin (1.0 mM). The
experimental design was completely randomized with four
treatments. Each experimental unit was constituted by two Petri
dishes containing eight explants each replicated four times. The
rate of callus induction was scored after 6 weeks in culture. The
data were transformed to log(x + 2) and submitted to ANOVA
and to SNK test (5.0%) according to Compton (1994).
2.3. Regeneration
Yellow nodule clusters originated in the MS culture medium
supplemented with 2,4-D (20.0 mM) and Kin (1.0 mM), were
selectively transferred to test tubes containing 15 ml of MS
culture medium supplemented with: (1) BAP (2.5 mM) + NAA
(0.5 mM); (2) Kin (2.5 mM) + NAA (0.5 mM); (3) 2-iP
(2.5 mM) + NAA (0.5 mM); (4) MS free of PGR. Each
experimental unit was constituted by two test tubes containing
0.25 g fresh weight of these yellow-greenish compact clusters
replicated five times in a completely randomized experimental
design. Data of bud regeneration and rooting were scored after
4 weeks in culture.
2.4. Histological analysis
Representative samples of different morphogenetic stages
were fixed in FAA–50 (Sass, 1951), dehydrated in an ascending
ethanol and xylol series, and included in paraffin. Serial
sections (8–12 mm) were obtained using a rotary microtome
Slee Technik1 and stained with safranin and fast-green.
Relevant aspects were identified and photographed using an
Olympus1 PM 20 camera coupled to an Olympus1 stereomicroscope SZH-10 and BX-40 microscope.
3. Results and discussion
3.1. Induction
Yellow nodule clusters similar to those described by Ziv
et al. (1995) were induced at a rate of 90.6% starting from the
explants cultured in MS culture medium supplemented with
20.0 mM 2,4-D plus 1.0 mM of Kin, after 6 weeks in culture
(Table 1). These nodule clusters were globular in form,
translucent to yellowish in color, and compact in texture, and
were originated arising from the basal region of the explants
(Fig. 1a). More specifically, the development of these nodule
clusters was observed starting from the parenchimatic tissues of
explants after 3 weeks in culture (Fig. 1b and d). The high
regenerative potential of leaf explants was ascribed to the
presence of intercalary axillar meristem in the basal region of
bromeliad leaves (Hosoki and Asahira, 1980).
The isolation and the subculture of the yellow nodule
clusters every 4 weeks in MS medium supplemented with 2,4-D
(5.0 mM) and Kin (0.5 mM) allowed their establishment and
multiplication (Fig. 1c). The characterization of these nodules
as cell agglomerates with consistent patterns of histodifferentiation was reported in the tissue culture of several
herbaceous and woody species like eucalyptus and pineapple
(Teng, 1997).
The nodule clusters showed the presence of vascular bundles
(Fig. 1d). Batista et al. (2000) showed the presence of
polycenter nodules formed from green organogenic nodular
cluster in Humulus lupulus. Morphogenic structures with high
regenerative potential and similar to those described in the
present work were observed in the tissue culture of the
Brazilian endemic bromeliads Vriesea friburgensis var.
paludosa (Alves and Guerra, 2001), Vriesea fosteriana
(Mercier and Kerbauy, 1997) and V. reitzii (Rech Filho
et al., 2005). Thus, it is possible that this morphogenic pattern is
recurrent in the genus Vriesea and a common feature in the
Bromeliaceae family. In Dyckia macedoi structures similar to
Table 1
Induction of yellow nodule clusters in Vriesea reitzii leaves cultured in MS
medium supplemented with 2,4-D and Kin (1 mM), after 6 weeks in culture
Plant growth regulators (mM)
% Induction
2,4-D (20.0) + Kin (1.0)
2,4-D (10.0) + Kin (1.0)
2,4-D (5.0) + Kin (1.0)
2,4-D (0) + Kin (0)
Mean
CV (%)
90.6
67.2
45.3
6.2
52.3
14.0
A
B
B
C
Values followed by different letters indicating significant differences according
to the SNK test ( p = 0.05). Data transformed for analysis using log(x + 2).
206
G.M. Alves et al. / Scientia Horticulturae 110 (2006) 204–207
Fig. 1. (a) Yellow nodule clusters induced from the explants after 3 weeks in culture (bar = 0.88 mm); (b) longitudinal section of explants showing meristematic
sectors from which nodule clusters arose (see arrow, bar = 0.176 mm); (c) nodule cultures maintained in MS culture medium supplemented with 2,4-D (5 mM) and
Kin (0.5 mM) (bar = 1.173 mm); (d) longitudinal section of nodule clusters showing the presence of vascular bundles (see arrow, bar = 66.7 mm); (e) adventitious
buds arising from nodule clusters in MS culture medium supplemented with 2-iP (2.5 mM) and NAA (0.5 mM) (bar = 0.587 mm); (f) longitudinal section of an
adventitious bud showing the apical meristem (see arrow, bar = 0.176 mm); (g) clusters of shoot-buds after 6 weeks in culture (bar = 0.88 mm); (e and h) adventitious
shoots after 8 weeks in culture (bar = 0.88 mm); (i) acclimatized plantlets (bar = 1.0 cm).
those described in the present work and presenting high
regenerative competence were originated from leaf explants
(Mercier and Kerbauy, 1992, 1993). In Neoregelia cruenta the
protuberances were also originated from the basal region of
leaves cultured in MS medium supplemented with 2.5 mM NAA
and 22.0 mM BA. This is relevant since the in vitro morphogenesis in bromeliads is normally associated to organogenesis based
on the release of axillary buds (Carneiro et al., 1999).
In V. reitzii the induction of protuberances and the
subsequent development of buds in MS culture medium
supplemented with NAA and BA allowed the regeneration of
plantlets for conservation purposes (Rech Filho et al., 2005). In
the present work the nodule clusters arose from the basal region
of individualized young leaves in response to the MS culture
medium supplemented with 2,4-D and Kin (Table 1). The
subsequent development of micro shoots occurred in the MS
culture medium supplemented with 2-iP and NAA.
3.2. Regeneration
The MS culture medium supplemented with 2.5 mM 2-iP
and 0.5 mM ANA resulted in 80.6% of shoot regeneration and
61.1% of shoot rooted after 4 weeks in culture (Table 2). The
estimated mean regeneration rate was 60.0 shoots/g fresh
weight of nodule clusters after 3 weeks in culture (Fig. 1e). In
H. lupulus the regeneration rate was 116 shoots/g callus after
one month in culture (Batista et al., 2000). Histological analysis
revealed the conspicuous development of the apical meristem
G.M. Alves et al. / Scientia Horticulturae 110 (2006) 204–207
Table 2
Regeneration of shoots and rooting from nodule cluster of V. reitzii cultured in
MS medium supplemented with Kin, BA and 2-iP (2.5 mM each) and NAA
(0.5 mM) after 4 weeks in culture
Plant growth regulators (mM)
% Shoot regeneration
% Rooting
2-iP (2.5 mM) + NAA (0.5 mM)
BAP (2.5 mM) + NAA (0.5 mM)
Kin 2-iP (2.5 mM) + NAA (0.5 mM)
MS PGR free
Mean
CV (%)
80.6
69.4
61.1
61.1
68.1
16.0
61.1
38.9
50.0
38.9
47.2
40.0
a
a
a
a
a
a
a
a
Mean followed by the same letter are not statistically different according to the
SNK test ( p = 0.05).
of an adventitious bud (Fig. 1f) after 4 weeks in MS culture
medium supplemented with 2.5 mM 2-iP and 0.5 mM NAA.
The morphogenetic pattern observed in the present work
resembles some structural features observed in the somatic
embryogenesis pathway, mainly associated with the development of structures with meristematic tissues (Fig. 1f). Similarly,
in the tissue culture of orchids the pattern of regeneration is
associated with the induction and development of structures
resembling protocormoids (George, 1993).
In the present work the subculture of nodule clusters to MS
medium supplemented with 2.5 mM 2-iP and 0.5 mM NAA
promoted the development of shoot-buds of H. lupulus as
described by Batista et al. (2000) after 6 weeks in culture
(Fig. 1g). The subculture of these micro shoots to MS PGR free
medium resulted in their elongation (Fig. 1h). Plantlets longer
than 3.0 cm obtained in such conditions were transferred to
trays containing substrate composed of 1:1 (v/v) of carbonized
rice coat and Turfa Fertil1 mineral supplement in a
nebulization tunnel. After 60 days the survival rate was 90%
(Fig. 1i).
Visual and anatomic observations did not detect any
morphologic alteration after acclimatization possibly attributed
to somaclonal variation. Future works should include the
assessment of genotypic fidelity as performed in the bromeliad
Ananas comosus by Feuser et al. (2003).
In conclusion the results of the present work showed the
competence of the basal region of leaves of V. reitzii for the
long-term regeneration and multiplication of nodule clusters.
The subculture of these structures to MS media supplemented
with 2.5 mM 2-iP and 0.5 mM NAA and subsequently to MS
PGR free allowed the development of shoot-buds and the
subsequent development of plantlets. The mean regenerative
rate was considerably higher than the values normally reported
in other in vitro regenerative protocols.
The in vitro morphogenetic patterns described may be
successfully employed for the mass propagation of endangered
bromeliads that also have ornamental value, as is the case of V.
reitzii.
207
Acknowledgements
This work was supported by CAPES, CNPq, and FAPESP.
References
Alves, G.M., Rech Filho, A., Puchalski, A., Reis, M.S., Nodari, R.O., 2004.
Allozymic markers and genetic characterization of a natural population of
Vriesea friburgensis var. paludosa, a bromeliad from the Atlantic Forest.
Plant Gen. Resources 2, 23–28.
Alves, G.M., Guerra, M.P., 2001. Micropropagation for mass propagation and
conservation of Vriesea friburgensis var. paludosa from microbuds. J.
Bromeliad Soc. 51, 202–212.
Batista, D., Ascensão, L., Sousa, M.J., Pais, M.S., 2000. Adventitious shoot
mass production of hop (Humulus lupulus L.) var. Eroica in liquid medium
from organogenic nodule cultures. Plant Sci. 151, 47–57.
Carneiro, L.A., Araujo, R.F.G., Brito, G.J.M., Fonseca, M.H.P.B., Costa, A.,
Crocomo, O.J., Mansur, E., 1999. In vitro regeneration from leaf explants of
Neoregelia cruenta (R. Graham) L.B. Smith, an endemic bromeliad from
Eastern Brazil. Plant Cell Tissue Org. Cult. 55, 79–83.
Compton, M., 1994. Statistical methods suitable for the analysis of plant tissue
culture data. Plant Cell Tissue Org. Cult. 37, 217–242.
Feuser, S., Meler, K., Daquinta, M., Guerra, M.P., Nodari, R.O., 2003. Genotypic fidelity of micropropagated pineapple (Ananas comosus) plantlets
assessed by isozyme and RAPD markers. Plant Cell Tissue Org. Cult. 72,
221–227.
George, E.F., 1993. Plant propagation by tissue culture: the technology. Part 1, 2
ed. Exegetics, Edington, 574 pp.
Hosoki, T., Asahira, T., 1980. In vitro propagation of bromeliads in liquid
culture. HortScience 15, 603–604.
Leme, E.M.V., Costa, A., 1991. A new species from Southern Brazil, a tribute to
Father Raulino Reitz. J. Bromeliad Soc. 41, 195–198.
Mercier, H., Kerbauy, G.B., 1992. In vitro multiplication of Vriesea fosteriana.
Plant Cell Tissue Org. Cult. 30, 247–249.
Mercier, H., Kerbauy, G.B., 1993. Micropropagation of Dychia macedoi—an
endangered endemic Brazilian bromeliad. Botanic Gardens Micropropagation News 1, 70–72.
Mercier, H., Kerbauy, G.B., 1997. Micropropagation of ornamental bromeliads
(Bromeliaceae). Biotechnol. Agric. For. 40, 43–57.
Morel, G.M., Wetmore, R.H., 1951. Tissue culture of monocotyledons. Am. J.
Bot. 38, 138–140.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and biossays
with tobacco tissue cultures. Physiol. Plant 15, 473–497.
Pompelli, M., Guerra, M.P., 2004. Ex situ conservation of Dickia distachia, an
endangered bromeliad from South Brazil. Crop Breed. Appl. Biotechnol. 4,
273–279.
Rech Filho, A., Dal Vesco, L.L., Nodari, R.O., Lischka, R.W., Müller, C.V.,
Guerra, M.P., 2005. Tissue culture for the conservation and mass propagation of Vriesea reitzii Leme and Costa, a bromeliad threatened of
extinction from the Brazilian Atlantic Forest. Biodiver. Conserv. 14 (8),
1799–1808.
Reitz, R., 1983. Bromeliáceas e a malária - bromélia endêmica. Flora ilustrada
Catarinense. 559 p.
Sass, I.E., 1951. Botanical Microtechnique. The Iowa State Coll. Press, New
York.
Teng, W.L., 1997. An alternative propagation method of Ananas comosus
trough nodule culture. Plant Cell Rep. 16, 454–457.
Ziv, M., Kahany, S., Lilien-Kipnis, H., 1995. Somatic embryos and bulblet
development from bioreactor regenerated meristematic clusters of nerine.
Acta Hortic. 393, 203–212.
Lirio Luiz Dal Vesco