Micropropagation of Juniperus phoenicea from adult plant explants and analysis of ploidy stability using flow cytometry
ABSTRACT We report here the successful micropropagation of adult Juniperus phoenicea L. with respective ploidy stability studies. Microcuttings with axillary buds were grown on five media supplemented with different growth regulator combinations. Best elongation rates were achieved on Driver and Kuniyuki (DKW) medium supplemented with kinetin alone or with naphthaleneacetic acid (NAA), while Rugini olive (OM) medium stimulated the development of new branches. Shoots growing on Murashige and Skoog (MS) medium browned and showed necrotic zones. Shoots of second to fourth subcultures usually had higher elongation rates than those of the first culture. For rooting assays, half strength DKW and OM media, different concentrations of growth regulators, auxin continuous exposure vs. dipping and the type of solid matrix were assessed. During rooting assays, two morphotypes were observed with one type having well developed internodes and the other showing hyperhydratation and no internode development. High rooting rates (40 %) were only obtained in the first morphotype shoots exposed for 5 min to 2.4 µM IBA and then transferred to OM medium without growth regulators. Plants were acclimatized in pots containing a mixture of peat and Perlite (3:2) in greenhouse with progressive reduction of relative humidity. A flow cytometric screening for major ploidy changes revealed no differences among the morphotypes and between them and the mother plant. Also the nuclear DNA content of this species was estimated for the first time using flow cytometry (2C = 24.71 pg). Additional key words: in vitro culture, nuclear DNA content, plant acclimatization, rooting studies.
- SourceAvailable from: José Pedro Fernandes[Show abstract] [Hide abstract]
ABSTRACT: We describe an encapsulation and dehydration procedure for the cryopreservation of cork oak (Quercus suber L.) somatic embryos that resulted in at least 90% survival. Genetic stability of the regenerated material was assessed by flow cytometry (FCM), amplified fragment length polymorphisms (AFLP) and simple sequence repeats (SSR). Cryopreservation of embryogenic clusters involved encapsulation of each cluster in an alginate bead, followed by a 3-day culture in 0.7 M sucrose and subsequent desiccation to 25 or 35% water content (WC), followed by freezing in liquid nitrogen. Thawed, cryopreserved somatic embryos had high viability and exhibited long-term survival. No morphological differences were observed between somatic embryos desiccated to 25 and 35% WC. Analysis of DNA ploidy stability of control (i.e., encapsulated and dehydrated but not frozen) and cryopreserved material by flow cytometry showed no significant differences. Similarly, DNA-marker analyses (AFLPs and SSR) revealed no significant differences between control and cryopreserved samples at the DNA-sequence level. Nonetheless, because polymorphisms were found between control material and samples cryopreserved and desiccated to 25% WC, the 35% WC method is recommended for cryopreservation of this tissue type. Cryopreservation of Q. suber somatic embryos by this encapsulation-dehydration procedure has potential for use in long-term conservation programs.Tree Physiology 01/2009; 28(12):1841-50. · 2.85 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Mediterranean junipers are of special ecological importance as key components of resource islands in semi-arid mountain ecosystems of the Mediterranean basin. The fragmentation of their habitat, which was primarily natural and driven by climatic drought conditions, is currently being aggravated by anthropogenic pressure. In the framework of this concern, the present work aims to contribute establishing a genomic profile of Juniperus in its western Mediterranean range, with a special emphasis placed on J. thurifera. DNA contents were assessed by flow cytometry in 43 populations of nine taxa within their Mediterranean range (first reports for J. navicularis, J. thurifera subsp. africana and J. thurifera subsp. thurifera). Chromosome numbers were determined by orcein staining in eight taxa (first counts for J. oxycedrus subsp. badia, J. phoenicea subsp. phoenicea, J. phoenicea subsp. turbinata, of 2n = 2x = 22, and for J. thurifera subsp. thurifera, of 2n = 4x = 44). Tetraploid cytotypes have been the only ones found in the 19 populations of J. thurifera studied, this being the first report of a Juniperus species exclusively polyploid. In J. thurifera, C-value does not respond to habitat fragmentation, in the same way that genetic diversity within populations was previously shown to be unaltered, suggesting that this factor has not had, at least to date, a significant impact on populations at genomic and genetic levels. Habitat fragmentation leads to deeply age-biased populations with a male-biased imbalanced sex ratio (lack of females), indicating an urgent need to improve regeneration within the populations of this species.Tree Genetics & Genomes 9(2). · 2.40 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Due to their position of interface between the sea and land, the flora and vegetation of coastal beaches and dunes, occupy ecologically extreme, sensitive, unique and valuable habitats. The occurrence of a large number of endemic taxa and specific plant associations endowed with key ecological services and adapted to a stressful and harsh environmental gradient, gives them a high interest for nature conservation and an important role in sustainable territorial planning. However, such ecosystems are vulnerable to the disruption caused by several anthropogenic sources. Among other global threat factors, the inevitable sea rise caused by climate change and, at a local scale, the non-negligible implications of trampling caused by disorderly coastal touristic exploitation, growing construction pressure in the littoral, and a seasonal population boom in late spring and in summer, plus all derivate forms of pollution, are threat factors to their integrity. Therefore, a correct planning of the touristic economic activities requires the understanding of the vegetation composition and spatial distribution patterns, intrinsically determined by their biogeographic context in the Euro-Siberian or Mediterranean Regions. This comprehensive work, based on a broad phytogeographic study, brings together disperse information on plant communities of the Portuguese sandy coasts (beaches and dunes), by analysing floristic compositions, chorology and ecological characteristics, and matching them with the “Nature 2000” network habitats. Resilience and vulnerability are also studied. In a nature conservation perspective, a positive balance (and a sustainable co-existence) between the preservation of natural values and human development in the Portuguese coast, will benefit with the integration of this knowledge in coastal planning and management activities.Journal of Coastal Conservation 03/2014; 17(3):279-299.
BIOLOGIA PLANTARUM 51 (1): 7-14, 2007
Micropropagation of Juniperus phoenicea from adult plant explants
and analysis of ploidy stability using flow cytometry
J. LOUREIRO, A. CAPELO, G. BRITO, E. RODRIGUEZ, S. SILVA, G. PINTO and C. SANTOS1
Laboratory of Biotechnology and Cytomics, Department of Biology, University of Aveiro,
Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
We report here the successful micropropagation of adult Juniperus phoenicea L. with respective ploidy stability studies.
Microcuttings with axillary buds were grown on five media supplemented with different growth regulator combinations.
Best elongation rates were achieved on Driver and Kuniyuki (DKW) medium supplemented with kinetin alone or with
naphthaleneacetic acid (NAA), while Rugini olive (OM) medium stimulated the development of new branches. Shoots
growing on Murashige and Skoog (MS) medium browned and showed necrotic zones. Shoots of second to fourth
subcultures usually had higher elongation rates than those of the first culture. For rooting assays, half strength DKW
and OM media, different concentrations of growth regulators, auxin continuous exposure vs. dipping and the type of
solid matrix were assessed. During rooting assays, two morphotypes were observed with one type having well
developed internodes and the other showing hyperhydratation and no internode development. High rooting rates (40 %)
were only obtained in the first morphotype shoots exposed for 5 min to 2.4 µM IBA and then transferred to OM
medium without growth regulators. Plants were acclimatized in pots containing a mixture of peat and Perlite (3:2) in
greenhouse with progressive reduction of relative humidity. A flow cytometric screening for major ploidy changes
revealed no differences among the morphotypes and between them and the mother plant. Also the nuclear DNA content
of this species was estimated for the first time using flow cytometry (2C = 24.71 pg).
Additional key words: in vitro culture, nuclear DNA content, plant acclimatization, rooting studies.
The genus Juniperus has about 75 to 80 species and is the
second largest genus of conifers. Juniperus species are
widely distributed in semiarid regions where they are
used extensively for landscaping, wood and medicinal
purposes. Juniperus phoenicea L. is native to some
regions of the Mediterranean basin, Canary Islands and
North Africa. Recently the taxonomic position of this
species was questioned (for details see Rivas-Martínez
et al. 1993) and this misleading taxonomy data requires
The lack of pollination, low pollen viability and/or
Received 23 March 2005, accepted 20 February 2006.
Abbreviations: 2,4-D - 2,4-dichlorophenoxyacetic acid; BAP - benzylaminopurine; DKW - Driver and Kuniyuki medium;
FCM - flow cytometry; IAA - indole-3-acetic acid; IBA - indole-3-butyric acid; KIN - kinetin; MS - Murashige and Skoog medium;
NAA - naphthaleneacetic acid; OM - Rugini olive medium; PI - propidium iodide; SH - Schenk and Hildebrandt medium;
TRIS - 3-(hydroxymethyl)-aminomethane; WPM - McCown Woody plant medium.
Acknowledgements: Conceição Santos is a collaborator of CESAM/UA. This work was supported by FCT project
PNAT/1999/AGR/15011/C. The acquisition of the flow cytometer was supported by the FCT/MCT project POCTI/AGR/C/11142/98.
The work of João Loureiro in flow cytometry was financed by the fellowship FCT/SFRH/BD/9003/2002 and the work of Glória
Pinto was financed by the fellowship FCT/SFRH/BD/8693/2002. Thanks are due to Prof. Jaroslav Doležel for providing the plant
DNA standard seeds. Thanks are also due to Eng. Armando Costa for technical assistance in the laboratory and to Dr. Roberto Jardim
(Director of the Botanical Garden of Madeira) and Quinta das Palmeiras for providing some of the material used in this study.
1 Corresponding author; fax: (+351) 234426408, e-mail: firstname.lastname@example.org
embryo degeneration are important causes for reduced
seed production in Juniperus (Ortiz et al. 1998). Also,
species that grow upright are more recalcitrant than
prostrate ones (Hartman et al. 1990). Dirr and Heuser
(1987) reported that cuttings of some Juniperus species
could be rooted using up to 4.5 % (m/v) indole-3-butyric
acid (IBA). Also, Hartman et al. (1999) reported
propagation of J. virginiana and J. procumbens by
cuttings and of J. virginiana and J. chinensis by seed
germination, but highlighted that the success of rooting
may be lower than 10 % when adult trees are
J. LOUREIRO et al.
used as mother plants (Edson et al. 1996). Unfortunately,
J. phoenicea is not efficiently propagated by traditional
methods and results are extremely inconsistent and not
reproducible (Brito 2000). For example, and similarly to
other Juniperus species, J. phoenicea does not have a
high rate of plant production through seed germination
(Ortiz et al. 1998).
Therefore, micropropagation should be performed in
order to improve the propagation of Juniperus species.
Contrarily to Pinus (e.g. Gomez and Segura 1995, 1996,
Andersone and Ievinsh 2005) few information is
available in Juniperus species. Gomez and Segura (1995)
reported the proliferation of J. oxycedrus by axillary
shoot proliferation and later found some morphogenic
capacity in calluses derived from single cell culture of the
same species (Gomez and Segura 1996). More recently,
Shanjani (2003) highlighted the importance of nitrogen
on callus induction and plant regeneration of J. excelsa,
and recently new media formulation based on the explant
mineral composition are being proposed as a new strategy
for highly recalcitrant species (e.g. Gonçalves 2004).
Alternatively, a new method involving germination of
in vitro cultured embryos on MS medium was proposed
to improve germination and propagation of J. oxycedrus
ssp. oxycedrus and ssp. macrocarpa (Cantos et al. 1998).
Materials and methods
In vitro culture establishment: Cuttings (15 - 20 cm
long) from terminal branches of adult trees (20-year-old)
from Porto Santo Island, collected in the spring, were
used as source of microcuttings for in vitro studies. Half
of the cuttings were treated immediately and the other
half was preconditioned in a greenhouse (22 ± 1 ºC, 16-h
photoperiod with irradiance of 400 µmol m-2 s-1) for at
least one week with periodic sprays of a fungicide
solution: 0.75 g dm-3 Derosal (Hoescht and Schering
AgrEvo, Berlin, Germany) and 1.5 cm3 dm-3 PrevicurN
(Hoescht and Schering AgrEvo). Cuttings were washed in
tap water for 10 min, decontaminated in ethanol 70 %
(v/v) for 1 min and then immersed two times (10 min
each) in 200 cm3 of a commercial bleach solution (2.5 -
3.0 % available chloride) containing 5 drops of Teepol
(Cruz Verde, Lisboa, Portugal). Cuttings were then rinsed
in a sterilised fungicide solution of 1g dm-3 Benlate
(Rhône-Poulenc, Lisboa, Portugal) for 10 min and
washed in sterile water three times (5 min each).
To test the influence of medium composition and
growth regulators on shoot propagation, decontaminated
initial explants (1 cm tall cuttings with one axillary bud)
were placed on different agar media: DKW (Driver and
Kuniyuki 1984), MS (Murashige and Skoog 1962), OM
(Rugini 1984), SH (Schenk and Hildebrandt 1972) or
WPM (McCown and Lloyd 1981), supplemented with
different growth regulators (Table 1). Each treatment
consisted of ten 400 cm3 flasks (with 50 cm3 medium)
containing five explants each giving a total of 50 explants.
Due to the possible occurrence of somaclonal
variation the analysis of the ploidy stability of
micropropagated plants is of particular importance. These
types of studies in conifers are rare and the ones using
flow cytometry (FCM) are even rarer. Gajdošová et al.
(1995) used this technique to analyse the genetic stability
of embryogenic calli of silver fir and its hybrids and
Libiaková et al. (1995) analysed the genetic stability of
Abies concolor × Abies grandis calli and in vitro
regenerated shoots. FCM has the advantages that nuclei
are analysed individually and at high speed, large
populations can be measured in a short time and the
presence of subpopulations can be detected (Shapiro
2003). Also, since there is no need to employ tissues with
dividing cells and it is easy and rapid to prepare samples,
FCM became the preferred method to perform this type
of analyses (Doležel and Bartoš 2005).
This investigation describes for the first time a
reliable protocol for in vitro regeneration and
acclimatization of J. phoenicea plants derived from an
adult field tree. The ploidy stability of plantlets derived
from the micropropagation protocol and from rooting
assays was assessed using FCM and the nuclear DNA
content of this species was determined for the first time
using leaves of the mother field tree.
Cultures were incubated in a growth chamber at 22 ± 1 ºC,
with a 16-h photoperiod and irradiance of 98 µmol m-2 s-1
supplemented by OSRAM (Munich, Germany) L36W/21
The influence of culture cycle on shoot multiplication
was evaluated by comparing shoots response in different
culture periods: 1st culture (shoots derived from the field
mother plant), 2nd culture (shoots derived from 2-month
old shoots of the 1st culture) and so on.
Shoot survival and morphological characters as shoot
length, number of shoots per explant and number of
branches per shoot were evaluated in each culture period.
Callus production was also evaluated. Treatments were
repeated two times in independent experiments.
Rooting studies and plant acclimatization: Shoots
(2.0 - 3.0 cm long) were transferred to different rooting
conditions: various basal culture medium, type and
concentration of growth regulator, continues exposure to
auxin vs. auxin dipping for 1 min, 5 min or 1 h, and
composition of the solid matrix (Table 2). Cultures were
incubated under the conditions described previously for
shoot culture. Each treatment consisted of eight 400 cm3
flasks containing 5 explants each giving a total of
40 explants. When roots were at least 2 cm long, in vitro
plantlets were transferred to pots with sterilised mixture
of peat : Perlite (3:2) and treated with a fungicide
solution: 0.75 g dm-3 Derosal and 1.5 cm3 dm-3 PrevicurN.
After that, they were transferred to a greenhouse and
MICROPROPAGATION OF JUNIPERUS PHOENICEA
grown at 22 ± 1 ºC, with a 16-h photoperiod and an
irradiance of 400 µmol m-2 s-1, where they were
acclimatized to progressive decreasing of relative
Ploidy stability analysis using flow cytometry: Nuclear
suspensions from micropropagated and mother plant
leaves were prepared according to Galbraith et al. (1983).
In short, nuclei were released from cells by chopping the
tissue with a razor blade in a Petri dish containing 1 cm3
of Tris-MgCl2 buffer (Pfosser et al. 1995) (200 mM TRIS,
4 mM MgCl2 . 6 H2O, 0.5 % (v/v) Triton X-100, pH 7.5).
The buffer was supplemented with 1 % (m/v) PVP-10 to
reduce the possible influence of secondary metabolites on
propidium iodide (PI) staining. The suspension of nuclei
was then filtered through a 50 μm nylon filter and
50 μg dm-3 of PI (Fluka, Buchs, Switzerland) and
50 μg dm-3 of RNAse (Sigma, St. Louis, USA) were
added to the samples to stain the DNA. Samples were
analysed within a 10 min period in a Coulter EPICS XL
(Coulter Electronics, Hialeah, FL, USA) flow cytometer
equipped with an air-cooled argon-ion laser tuned at
15 mW and operating at 488 nm. Integral fluorescence
and fluorescence pulse height and width emitted from
nuclei were collected through a 645-dichroic long-pass
filter and a 620-band-pass filter and converted on
1 024 ADC channels. Prior to analysis, the instrument
was checked for linearity with Flow-Check fluorespheres
(Beckman Coulter, Hialeah, FL, USA). Doublets, partial
nuclei, nuclei with associated cytoplasm and other debris
were discriminated using a specific gating region defined
in a linear-fluorescence light scatter (FL) pulse integral vs.
FL pulse height cytogram. Leaves of Secale cereale
cv. Dankovske (2C = 15.95 pg, Doležel et al. 1998) were
used as an internal reference standard. At least
Results and discussion
In vitro culture establishment: Higher surviving rates
(58 % from a total of 30 explants) were obtained with
explants derived from preconditioned cuttings (that were
periodically treated with a fungicide solution) than with
explants derived directly from the field (22 % from a total
of 30 explants). The
preconditioning of field cuttings to prevent in vitro
infections was already applied in micropropagation
studies of other field trees with similar success (Pinto
et al. 2002). Simultaneously, the method used for
explants disinfection was successful.
Explants growing on DKW, OM and SH media
presented, in the first culture, similar surviving rates
(P ≤ 0.05) that were slightly higher than those of explants
growing on WPM (P ≤ 0.1; data not shown) and higher
than the surviving rates of explants on MS medium,
which was more evident during the 2nd to 4th subcultures.
In fact, with the exception of MS and SH media, survival
rates increased considerably in subsequent subcultures
5 000 nuclei were analysed per sample.
To estimate the ploidy level, the position of the G0/G1
peak of the sample on a histogram was compared with the
internal reference plant with known ploidy. The size of
the nuclear genome of J. phoenicea was estimated
according to the following formula:
J. phoenicea 2C nuclear DNA content [pg] =
(J. phoenicea G0/G1 peak mean / S. cereale G0/G1 peak
mean) × 15.95. Conversion into base-pair numbers was
performed using the following factor: 1 pg = 978 Mbp
(Doležel et al. 2003).
The relative nuclear DNA content of 4 micropropa-
gated plantlets obtained in OM medium was estimated
and compared with the values obtained from 5 replicates
of the adult mother field tree. To assay the possible
influence of different rooting conditions on explants
ploidy stability, the relative nuclear DNA content of at
least one explant in OM medium from each rooting
condition was estimated.
Statistical analysis: Data from in vitro culture and
rooting studies were averaged from two independent
analyses and were analysed using the one-way analysis of
variance (ANOVA). A multiple comparison Tukey-
Kramer test was applied when necessary to determine
exactly which groups were different (P < 0.05).
Statistical analyses of data from ploidy stability assays
were performed using an unpaired t-test (for comparison
between micropropagated plantlets and the adult mother
plant; P < 0.05) and a two-way ANOVA (for comparison
between different rooting conditions – auxinic dipping
and composition of solid matrix – and detection of
possible interactions between them; P < 0.05). All
statistical studies were performed using SigmaStat
(Windows Version 3.1, SPSS Inc., Richmond, USA).
strategy of greenhouse
(76 - 100 %). On MS medium, shoots browned and
showed apical necrosis that eventually spread during
subcultures to the whole shoot leading to its death.
Therefore, this medium was not suitable to establish in
vitro shoot cultures in this species (Fig. 1A). On SH
medium some of shoots dying, while the surviving shoots
showed a light green colour and new growing zones.
No significant differences were obtained among
different growth regulator combinations in regards of
shoot survival rates; 100 % survival rates were obtained
on DKW medium with 0.93 µM kinetin (KIN) or with
0.45 µM BAP and on OM medium with 2.74 µM NAA
and 1.86 µM KIN.
Independently of the subculture, some morphologic
differences (shoot length, number of shoots per explant
and number of branches per shoot) were observed. Shoots
grown on MS medium showed the lowest survival,
lowest number of shoots per explant and a reduced
number of branches per shoot (P ≤ 0.05; Table 1). Shoots
J. LOUREIRO et al.
grown on WPM also had low number of shoots per
explants and low branching, but elongation was in
general higher than on MS medium (P ≤ 0.05) and
oxidation was lower than on MS (Table 1). On DKW
medium explants had higher elongation rates when
compared to all the other media (P ≤ 0.05, Table 1),
presenting well developed branches and green leaves
(Fig. 1B). In this medium the number of shoots per
explant and the number of branches per shoot were
among the highest values obtained. Only on OM medium
better results were obtained concerning these two
characters (P ≤ 0.05). On this medium, high amounts of
tissue developed at the base of the shoots although
elongation was significantly lower than on DKW
Fig. 1. Juniperus phoenicea shoot culture on A - MS medium (arrows are pointing out necrotic regions, bar = 0.5 cm), B - DKW
medium (bar = 0.5 cm) and C - OM medium (bar = 0.5 cm). Shoot morphotypes were observed during the same rooting conditions
(shoots exposed for 5 min to 2.4 µM IBA and transferred to OM medium without growth regulators): D - morphotype 1 with well
developed branches and green leaves (bar = 0.5 cm), E - morphotype 2 with high vitrification, short internodes and light green leaves
(bar = 0.5 cm), F - 4-month-old morphotype I plantlet with well developed roots (bar = 0.5 cm); G,H - plantlets during
acclimatization on peat : Perlite (3:2) in greenhouse for 3 d (G) (bar = 1.0 cm) or 3 months (H) (arrows are pointing out new leaves,
bar = 0.5 cm). I - typical callus formed on OM medium (see root development) (bar = 1.0 cm).
MICROPROPAGATION OF JUNIPERUS PHOENICEA
Table 1. Effect of basal media and growth regulator combinations on Juniperus phoenicea survival rate, mean shoot length and
number of shoots and number of formed branches during 1st culture and at the end of 4th subculture (mean shoot length at the
beginning of every culture = 1.3 ± 0.3 cm). In each parameter the same letter (a, b or c) means that no significant differences were
detected according to the one-way analysis of variance and to the multiple comparison Tukey-Kramer test (P ≤ 0.05; from two
independent experiments, n = 30). Last column refers to the capacity of different media to produce and maintain calli (- no
production; + low production; ++ good production). nd - not determined.
Medium Growth regulators Shoot survival
Number of shoots
Number of branches
[µM] 4th 4th 4th 4th
2.74 NAA + 1.86 KIN 63±11a 93± 2b 4.8±0.6c 6.1±0.8c 5.3±2.5bc 3.3±1.3b
2.74 NAA + 0.45 BAP 70±14a 90± 6b 3.8±0.8ab 3.8±0.8b 4.1±1.0b 4.2±0.9b
2.74 NAA + 0.90 BAP 68±15a 92± 4b 4.0±0.4b 4.1±0.2b 4.8±1.1b 4.8±1.1b
60±18a 66± 2a
65±16a 94± 2b 3.2±0.1ab 3.4±0.2ab 8.3±1.8c 8.3±1.8c
70±21a 90± 7b 2.2±0.5a 3.1±0.1ab 7.2±2.0c 7.6±2.0c
2.74 NAA + 1.86 KIN 72±10a 100± 0c
2.74 NAA + 0.45 BAP 65±12a 95± 2b 2.6±0.2a 3.0±0.7ab 8.5±0.5c 8.5±0.5c
2.74 NAA + 0.90 BAP 72±8a 90± 4b 2.8±0.5a 2.9±0.3ab 6.2±0.3bc 6.2±0.3bc 54.2±11.3c 45.6± 9.6bc ++
58±15a 45±12a 2.1±1.0a nd
70±10a 61± 2a 1.0±0.8a 2.3±0.2a 0.5±0.2a 1.4±0.23a nd
64±20a 85± 6b 3.8±0.4b 4.3±0.5b 2.7±0.6ab 1.1±0.2a
65±17a 90±10b 3.4±0.5ab 3.8±0.6b 2.1±0.3ab 2.1±1.0a
2.74 NAA + 0.86 KIN 70±22a 90± 0b 2.0±0.2a 2.8±0.1a 2.8±0.4b 2.3±0.8ab 14.9±12.9a 21.2± 5.9a -
2.74 NAA + 0.45 BAP 67±10a 88± 6b 2.6±0.6a 2.9±0.6a 2.5±0.4ab 1.7±1.1a
2.74 NAA + 0.90 BAP 62±24a 76±10b 3.4±0.5ab 3.9±0.5bc 2.5±0.8b 1.5±1.3a
70±13a 100± 0c
72±10a 100± 0c
4.5±0.8bc 5.4±0.5bc 4.2±0.8b 4.5±1.2b
3.5±0.5ab 4.8±0.3b 4.7±1.3b 3.1±0.7b
27.4± 7.1b 32.6±14.2b -
23.7± 9.4b 35.4±18.4b -
39.1±10.5b 38.4±18.4b +
24.0±11.8b 39.6±15.7b -
22.4± 6.0b 37.6±11.1b -
18.6±14.2ab 15.4± 6.4a -
11.7± 7.3a 19.3± 8.5a -
43.0±17.5bc 45.4±10.1bc -
52.2±20.1c 61.6± 2.4c -
49.5±12.1c 59.3±21.7c ++
38.7±11.6bc 33.4±13.5b ++
2.5±0.9a 2.7±0.9a 1.0±0.0a 1.1±0.3a
2.1±0.6a 2.8±1.1a 1.1±0.2a 1.3±0.6a
3.0±0.4ab 4.0±0.5ab 9.4±3.0c 9.4±3.0c
nd nd nd nd
21.6± 7.3a 17.3± 6.6a -
19.7± 7.1a 15.8± 3.4a -
16.5± 5.8a 11.1± 3.5a -
11.3± 4.7a 19.6± 5.2a -
Table 2. Effects of medium (½ DKW or OM), different concentrations of growth regulators (IAA, IBA or NAA), type of exposure to
growth regulators (continuous, 1 or 5 min dipping or 1 h exposure) and composition of solid matrix (agar or peat:Perlite; 3:2) on
rooting of Juniperus phoenicea explants (n = 40). In each parameter the same letter means that no significant differences were
detected (P < 0.05).
Medium Growth regulator
Exposure Solid matrix Rooting
Number of roots
1 min dipping
1 h exposure
1 min dipping
5 min dipping
1 min dipping
5 min dipping
(P ≤ 0.05). The most outstanding feature of shoots
developed on OM medium is that they looked completely
different from shoots developed on other media:
internodes were almost absent, and shoots had a high
amount of branches with lots of small, rolled, and dark
green leaves (Fig. 1C).
In J. oxicedrus the best medium for culture establish-
ment was a modified SH medium without growth
regulators or supplemented with BAP (Gomez and
Segura 1995). This medium was therefore tested in our
studies to micropropagate J. phoenicea. However, in this
species SH medium led to heterogeneous response with
some shoots dying during subsequent subcultures (Table 1).
Concerning the different growth regulator combina-
tions tested no significant differences (P ≤ 0.05, Table 1)
were detected in most cases. An exception was observed
in DKW medium where the combination of 2.74 µM
NAA and 1.86 µM KIN produced the best elongation
rates and a good number of shoots per explant and
branches per shoot.
J. LOUREIRO et al.
In general higher shoot lengths and number of
branches per shoot were observed during subsequent
subcultures. This may be explained by the fact that,
during 1st culture, growth could be limited by stress
imposed by the decontamination process and that shoots
need to adjust to the in vitro conditions as they derived
from stems of field plants. By other way, shoots in the 2nd
to 4th subcultures were already adapted and able to
maximise growth by using the conditions supplied by the
in vitro culture, and frequently, new formed regions
(evident by their light green colour) could be observed at
the top of the branches.
Rooting studies and plant acclimatization: For rooting
experiments two strategies were adopted concerning
shoots exposure to auxins (IAA, IBA and NAA):
continuous exposure or dipping for 1 min, 5 min or 1 h
(Table 2). These studies were performed on half strength
DKW (½ DKW) and OM medium as these were the
media where the best results were obtained previously.
On ½ DKW medium, rooting was only observed when
IBA was chosen. For this reason IBA was the growth
regulator in the OM medium rooting assays. Concerning
the continuous auxin exposure, rooting was only
observed on ½ DKW medium with 4.1 µM IBA (Table 2).
On this medium root primordia were observed in 6 % of
shoots and 4 weeks after transfer to rooting medium
(Table 2). When shoots were exposed for 1 h to 2.5 mM
IBA, roots appeared in 14 % of them and a 1 min dipping
induced roots in 6 % of the shoots.
On OM medium, dipping in 2.4 µM IBA induced
higher percentages of rooting (10 % for 1 min exposure
and 40 % for 5 min exposure). These values were higher
in agar medium when compared to the same conditions
on a peat and Perlite mixture (n = 20).
Comparing with previous results in the same genus,
this species seems to have different rooting abilities. For
example, rooting of J. scoppulorum reached 82 % (Dirr
and Heuser 1987). However, this macropropagation
depends on the species/ecotype, and is reduced when
adult trees are used. Wagner et al. (1994) obtained less
than 10 % rooting in cuttings from 12-year-old mother
plants. Gomez and Segura (1995) reported low rooting
capacity of in vitro shoots of J. oxycedrus, and the
genotypes of J. phoenicea from Porto Santo Island were
for a long time extremely recalcitrant to both ex vitro and
in vitro rooting (Brito 2000). Some of the reasons for this
difficulty may be the peculiar environmental conditions
of the Island together with the genotype/ecotypes used.
Finally, two morphotypes were observed: one group
(app. 70 %) defined as “Morphotype I” had apparently
normal shoots with green leaves, well developed
internodes and a large number of branches (Fig. 1D); the
other group (app. 30 %), defined as “Morphotype II”, was
characterized by frequent
short/almost absent internodes (Fig. 1E). Rooting was
only achieved in shoots of the “Morphotype I“ (Fig. 1F).
Plant acclimatization in greenhouse is still in course
and restricted to few plants but, in “Morphotype I“, the
acclimatisation is up to the moment 70 % successful
When developing a plant regeneration protocol, callus
formation should be avoided. However, among the
19 media combinations tested some led to the production
of callus tissue. This is the first report on callus
production in J. phoenicea (Fig. 1I). Callus production
may be important for studies of indirect morphogenesis
(e.g. somatic embryogenesis) – that are presently
occurring in our laboratory – or for studies of production
of secondary metabolites. An interesting example is the
production of high contents of podophyllotoxin (a strong
anti-tumor agent) in callus tissue from leaves of
J. chinensis on SH medium supplemented with 16.2 µM
NAA and 0.93 µM KIN (Muranaka et al. 1998). Other
growth regulator combinations as 4.5 µM 2,4-dichloro-
phenoxyacetic acid (2,4-D) and 0.93 µM KIN or 5.4 µM
NAA and 0.93 µM KIN also originated good calli
production although not so efficiently (Muranaka et al.
1998). Also, Gomez and Segura (1995, 1996) achieved
callus formation from adult J. oxycedrus plants in SH
medium with 2,4-D. Our results with J. phoenicea
confirm that, similarly to J. chinensis and among the
combinations tested, NAA plays an important role on
callus production as the media that induced callus had
this growth regulator (Table 1). On the other hand, stems
showed to be, in this species, a good explant source for
Ploidy stability: FCM analyses were applied to study the
ploidy stability of J. phoenicea plantlets derived from the
micropropagation protocol and from rooting assays. In
J. phoenicea to assure that a sufficient number of nuclei
were obtained the amount of material per sample had to
Fig. 2. Histogram of relative fluorescence intensity (FL)
obtained after simultaneous analysis of nuclei isolated from
Secale cereale (2C = 15.95 pg DNA, as an internal reference
standard) and Juniperus phoenicea leaves of the adult mother
plant stained with propidium iodide (PI). Four peaks were
observed in the histogram: 1 - nuclei at G0/G1 phase of S.
cereale; 2 - nuclei at G0/G1 phase of J. phoenicea; 3 - nuclei at
G2 phase of S. cereale; 4 - nuclei at G2 phase of J. phoenicea.
Despite being a woody plant species, which are
recalcitrant to this type of analyses (Loureiro et al. 2005),
MICROPROPAGATION OF JUNIPERUS PHOENICEA
Table 3. Nuclear DNA content of micropropagated plantlets and
adult mother field tree from which they were obtained. Means
± SD, CV - coefficient of variation. The differences were not
significant at P ≤ 0.05; n = 4 for micropropagated plantlets and
5 for trees. For detail see Materials and methods.
Adult mother tree
Table 4. Nuclear DNA content of Juniperus phoenicea
morphotypes cultivated on OM medium and dipped in 5.0 µM
IBA for 1 or 5 min before rooting on agar or peat:Perlite (3:2).
Means ± SD, CV - coefficient of variation. The differences were
not significant at P ≤ 0.05; n = 1 - 3. For detail see Materials
the mean coefficient of variation (CV) values obtained
(Table 3 and 4) were below 5 that is considered the
acceptance criterion by Galbraith et al. (2002). As
expected, the internal standard S. cereale had a lower 2C
distribution (mean CV = 3.0 %; Fig. 2). The development
of a protocol that gives low CV values is of crucial
importance (this issue is fully addressed by Pinto et al.
2004) as it is a measure of the precision of the analysis.
Unfortunately, in many reports this information is not
provided and this difficult results interpretation. There is
usually a correlation between higher CV values and
higher background noise, especially in lower channel
Andersone, U., Ienvish, G.: In vitro regeneration of mature
Pinus sylvestris buds stored at freezing temperatures. - Biol.
Plant. 49: 281-284, 2005.
Brito G.: Micropropagação de duas espécies autóctones da Ilha
de Porto Santo (Olea europaea L. ssp. maderensis Lowe e
Juniperus phoenicea L.) e estudo da resposta de rebentos in
vitro a stress osmótico. - Master Thesis, University of
Aveiro, Aveiro 2000.
Cantos, M., Cuerva, J., Zarate, R., Troncoso, A.: Embryo rescue
and development of Juniperus oxycedrus subsp. oxycedrus
and macrocarpam seed. - Sci. Technol. 26: 193-198, 1998.
Dhillon, S.S.: DNA in tree species. - In: Bouga, J.M., Deuzan,
numbers (Emshwiller 2002). This applies to this case
where some background noise was observed. Despite
that, the FCM protocol established here for J. phoenicea
provided good results demonstrating its reliability and
potential use in other Juniperus species.
Considering the in vitro culture protocol used to
establish J. phoenicea, the mean nuclear DNA
fluorescence index (DI = 2CJ. phoenicea/2CS. cereale) obtained
for the micropropagated plantlets (DI = 1.533) was very
similar (P ≤ 0.05) to the one obtained for the mother field
plant (DI = 1.549). These results strongly indicate that the
micropropagation protocol described here apparently
does not induce major ploidy changes. Nevertheless, the
CV values obtained could mask the possible occurrence
of small differences in nuclear DNA content (aneuploidy
and DNA polymorphism) and therefore complementary
studies, such as chromosome counting and micro-
satellites, to evaluate this situation, are presently being
conducted in our laboratory.
To assay the possible influence of different rooting
conditions on explants ploidy stability, the relative
nuclear DNA content of plantlets from each morphotype
was assessed (Table 4). The DI values obtained for each
set of conditions were very similar (P ≤ 0.05). Also, no
statistically significant differences were detected between
the two morphotypes, revealing that the morphologic
differences did not reflect major genetic changes. Up to
the moment, all micropropagated plants that were
acclimatized showed a normal development. Never-
theless, more refine techniques (as microsatellites to
assess DNA mutations and DNA methylation to evaluate
the influence of epigenetic changes) are currently being
performed in our laboratory.
The determination of nuclear DNA content of
J. phoenicea L. in absolute units was performed using the
adult field tree and the obtained value was 2C = 24.71 ±
0.178 pg (1C genome size = 12 083 Mbp). This is the
first report for this species and the obtained value is
similar to the values obtained by Dhillon (1987) using
Feulgen staining and microspectrophotometry and
Hizume et al. (2001) using FCM for other Juniperus
species (J. virginiana, J. conferta and J. rigida), and as
reported above FCM may also be used as an important
tool for taxonomic studies in this genus.
D.J. (ed.): Cell and Tissue Culture in Forestry. Pp. 298-313.
Martinus Nijhoff, Dordrecht 1987.
Dirr, M.A., Heuser, C.W. (ed.): The Reference Manual of
Woody Plant Propagation: From Seed to Tissue Culture. -
Varsity Press, Athens 1987.
Doležel, J., Bartoš, J.: Plant DNA flow cytometry and
estimation of nuclear genome size. - Ann. Bot. 95: 99-110,
Doležel, J., Bartoš, J., Voglmayr, H., Greilhuber, J.: Nuclear
DNA content and genome size of trout and human. -
Cytometry Part A 51: 127-128, 2003.
Doležel, J., Greilhuber, J., Lucretti, S., Meister, A., Lysák, M.,
J. LOUREIRO et al.
Nardi, L., Obermayer, R.: Plant genome size estimation by
flow cytometry: inter-laboratory comparison. - Ann. Bot.
82: 17-26, 1998.
Driver, J.A., Kuniyuki, A.H.: In vitro propagation of paradox
walnut rootstock. - HortScience 19: 507-509, 1984.
Edson, J.L., Wenny, D.L., Dumroese, R.K., Leege-Brusven, A.:
Mass propagation of rocky mountain juniper from stem
cuttings. - Tree Planters’ Notes 47: 94-99, 1996.
Emshwiller, E.: Ploidy levels among species in the Oxalis
tuberosa 'Alliance' as inferred by flow cytometry. - Ann.
Bot. 89: 741-753, 2002.
Gajdošová, A., Vooková, B., Kormuťák, A., Libiaková, G.,
Doležel, J.: Induction, protein-composition and DNA-ploidy
level of embryogenic calli of silver fir and its hybrids. - Biol.
Plant. 37: 169-176, 1995.
Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M.,
Sharma, D.P., Firoozabady, E.: Rapid flow cytometric
analysis of the cell-cycle in intact plant-tissues. - Science
220: 1049-1051, 1983.
Galbraith, D.W., Lambert, G., Macas, J., Doležel, J.: Analysis
of nuclear DNA content and ploidy in higher plants. - In:
Robinson, J., Azmi, A., Tutois, S. (ed.): Current Protocols
in Cytometry. Pp. 7.6.1-7.6.15. John Wiley & Sons, New
Gomez, M.P., Segura, J.: Axillary shoot proliferation in cultures
of explants from mature Juniperus oxycedrus trees. - Tree
Physiol. 15: 625-628, 1995.
Gomez, M.P., Segura, J.: Morphogenesis in leaf and single-cell
cultures of mature Juniperus oxycedrus. - Tree Physiol. 16:
Gonçalves, S., Correia, P.J., Martins-Loução, M.A., Romano,
A.: A new medium for in vitro rooting of carob tree based
on leaf macronutrients concentrations. - Biol. Plant. 49:
Hartman, H.T., Kester, D.E., Davies, F.T. (ed.): Plant
Propagation: Principles and Practices. - Prentice Hall
Hartman, H.T., Kester, D.E., Davies, F.T., Geneve, R. (ed.):
Plant Propagation: Principles and Practices. - Prentice Hall
International, Englewood Cliffs 1999.
Hizume, M., Kondo, T., Shibata, F., Ishizuka, R.: Flow
cytometric determination of genome size in the
Sciadopityaceae. - Cytologia 66: 307-311, 2001.
Libiaková, G., Gajdošová, A., Vooková, B., Kubincová, M.,
Doležel, J.: Karyological study of Abies concolor × Abies
grandis calli and shoots regenerated in vitro. - Biologia 50:
Loureiro, J., Pinto, G., Lopes, T., Doležel, J., Santos, C.:
sensu stricto and
Assessment of genetic stability of Quercus suber L. somatic
embryogenesis process using flow cytometry. - Planta 221:
McCown, B.H., Lloyd, G.: Woody plant medium (WPM) - a
mineral nutrient formulation for microculture of woody
plant-species. - HortScience 16: 453-453, 1981.
Muranaka, T., Miyata, M., Ito, K., Tachibana, S.: Production of
podophyllotoxin in Juniperus chinensis callus cultures
treated with oligosaccharides and a biogenetic precursor. -
Phytochemistry 49: 491-496, 1998.
Murashige, T., Skoog, F.: A revised medium for rapid growth
and bioassays with tobacco tissue cultures. - Physiol. Plant.
15: 473-497, 1962.
Ortiz, P.L., Arista, M., Talavera, S.: Low reproductive success
in two subspecies of Juniperus oxycedrus L. - Int. J. Plant
Sci. 159: 843-847, 1998.
Pfosser, M., Amon, A., Lelley, T., Heberle-Bors, E.: Evaluation
of sensitivity of flow cytometry in detecting aneuploidy in
wheat using disomic and ditelosomic wheat-rye addition
lines. - Cytometry 21: 387-393, 1995.
Pinto, G., Loureiro, J., Lopes, T., Santos, C.: Analysis of the
genetic stability of Eucalyptus globulus Labill. somatic
embryos by flow cytometry. - Theor. appl. Genet. 109: 580-
Pinto, G., Valentim, H., Costa, A., Castro, S., Santos, C.:
Somatic embryogenesis in leaf callus from a mature
Quercus suber L. tree. - In vitro cell. dev. Biol. Plant. 38:
Rivas-Martínez, S., Wildpret, W., Paz, P.L.P.: Datos sobre
Juniperus phoenicea aggr. (Cupressaceae). - Itinera Geobot.
7: 509-512, 1993.
Rugini, E.: In vitro propagation of some olive (Olea europaea
L. var. sativa) cultivars with different root-ability, and
medium development using analytical data from developing
shoots and embryos. - Sci. Hort. 24: 123-134, 1984.
Schenk, R.U., Hildebrandt, A.C.: Medium and techniques for
induction and growth
dicotyledonous plant-cell cultures. - Can. J. Bot. 50: 199,
Shanjani, P.S.: Nitrogen effect on callus induction and plant
regeneration of Juniperus excelsa. - Int. J. agr. Biol. 5: 419-
Shapiro, H.M. (ed.): Practical Flow Cytometry. - Wiley-Liss,
New York 2003.
Wagner, A.M., Harrington, J.T., Mexal, J.G., Fisher, J.T.:
Progress report on propagation of Juniperus for
conservation plantings. - In: Proceedings: Intermountain
Nursery Conference. Pp. 131-136. St. Louis 1994.
of monocotyledonous and