Trees (2013) 27:13391351

DOI 10.1007/s00468-013-0882-0

ORIGINAL PAPER

Somatic embryogenesis in Pinus halepensis Mill.: an important

ecological species from the Mediterranean forest
I. A. Montalban A. Setien-Olarra
C. L. Hargreaves P. Moncalean

Received: 31 January 2013 / Revised: 4 April 2013 / Accepted: 11 April 2013 / Published online: 1 May 2013
 Springer-Verlag Berlin Heidelberg 2013

Abstract Pinus halepensis Mill. is a common forest

species in the Mediterranean area and it is important for
environmental conservation. This study established a
method of regenerating Pinus halepensis Mill. through
somatic embryogenesis. The effect of culture medium
(mineral salts, nitrogen source and plant growth regulators), collection date and seed family on embryogenic tissue initiation and proliferation in Pinus halepensis was
analysed during the first steps of embryogenesis process.
This study showed a marked effect of the culture medium
tested as well as some significant differences among collection dates. Furthermore, the embryogenic tissue initiation was affected by the amino acid mixture in the culture
medium and the proliferation stage was significantly
affected by the combination of plant growth regulators. At
the end of the maturation phase the presence of activated
charcoal was also evaluated. Finally, maturation of
embryogenic tissue was affected by the nitrogen source in
the culture medium and these results were different for
high and low mature embryo producing cell lines. To the
best of our knowledge, this is the first report on Aleppo
pine somatic embryogenesis describing a simple and efficient procedure for large-scale somatic embryo production.
Keywords Aleppo pine
Embryogenic tissue

Germination
Initiation
Maturation
Propagation
Communicated by K. Klimaszewska.
I. A. Montalban
A. Setien-Olarra
P. Moncalean (&)
Centro de Arkaute, Neiker-Tecnalia, Apdo. 46,
01080 Vitoria-Gasteiz, Spain
e-mail: pmoncalean@neiker.net
C. L. Hargreaves
Scion, Private Bag 3020, Rotorua 3046, New Zealand

Abbreviations
A
Maturation medium, DCR basal medium
supplemented with: 75 lM ABA, 9 g L-1 of
Gelrite, 4.5 % (w/v) sucrose, and ED amino
acid mixture
ABA Abscisic acid
AC
Activated charcoal
B
Maturation medium, DCR medium supplemented
with: 75 lM ABA and 9 g L-1 of Gelrite, 4.5 %
(w/v) sucrose and ED amino acid mixture with
1,650 mg L-1 of glutamine
BA
N6-benzyladenine
C
Maturation medium, DCR basal medium
supplemented with: 75 lM ABA, 9 g L-1 of
Gelrite, 6 % (w/v) sucrose and ED amino acid
mixture
D
Maturation medium, DCR medium supplemented
with: 75 lM ABA and 9 g L-1 of Gelrite, 6 %
(w/v) sucrose and ED amino acid mixture with
1,650 mg L-1 glutamine
2,4-D 2,4-Dichlorophenoxyacetic acid
DBA 9 lM 2,4D and 2.7 lM BA
DCR Gupta and Durzan basal medium (Gupta and
Durzan 1985)
DKI
9 lM 2,4D and 2.7 lM Kinetin
DNB 4.5 lM 1-Naphthaleneacetic acid, 4.5 lM 2,4D
and 2.7 lM BA
ECLs Established cell lines
ED
EDM amino acid mixture
EDM Embryo development medium (Walter et al. 1998)
ET
Embryogenic tissue
FW
Fresh weight
LP
Quorin and Lepoivre medium (Quoirin and Lepoivre
1977, modified by Aitken-Christie et al. 1988)
SE
Somatic embryogenesis

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Introduction
Pinus halepensis Mill., commonly named Aleppo pine, is a
species native to the Mediterranean region and is widespread from Spain to Algeria (Botella et al. 2010). Temperature and precipitation requirements generally confine
its distribution to sub-humid areas of the Mediterranean. Its
main area of distribution is Southern Europe (Spain, Italy
and Greece) and Northern Africa (Algeria, Tunisia and
Morocco). In these regions, as described by Lambardi et al.
(1993), Aleppo pine is of a great economic importance due
to its adaptability to dry, calcareous and poor soils. In light
of predictions of global drying and warming for this region,
there is some concern about the physiological ability of P.
halepensis to persevere in large afforestations in the future
(Oliveras et al. 2003; Maestre and Cortina 2004). This
species is especially suitable for the reforestation of marginal and submarginal areas because it is one of the most
drought resistant pine species (Klein et al. 2011). Moreover, in marine stands, P. halepensis forests are important
both for defence against the saline winds and for landscape
purposes (Lambardi et al. 1993). In Spain, virtually all
natural stands are distributed over the whole Eastern coast,
though due to its important ecological plasticity it has also
been intensively used for afforestation in North-Western
areas of the Iberian Peninsula, very often out of its natural
habitat range (Abello 1998).
In vitro vegetative propagation from physiologically
juvenile tissue has been successful in a number of conifer
species (Moncalean et al. 2005). This phenomenon has led
many organizations to focus on production of elite families
through juvenile tissue propagation in the short-term, either
for operational use or for clonal tests, while they continue
to research methods for propagation of selected mature
trees (De Diego et al. 2008, 2010).
Conventional seed orchards provide genetically
improved seeds, but, as stated by Park et al. (1998) traditional breeding strategies combined with in vitro vegetative
propagation have shown advantages. These include additional genetic gain achieved by capturing non-additive
genetic variation and the capacity of introducing clones to
meet market goals at a higher speed. In this sense, propagation via somatic embryogenesis (SE) from immature
zygotic embryos is an effective method of propagation
when combined with other technologies, such as cryopreserving the embryogenic tissue (ET) and selecting elite
clones in field tests (Park 2002). This system offers the
capability to produce large numbers of somatic embryo
derived plantlets (Montalban et al. 2010) as well as to use
the somatic embryos for organogenesis procedures and thus
provide further amplification (Montalban et al. 2011a).
However, this technology has a number of bottlenecks in
the Pinus genus, namely, a narrow competence window for

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Trees (2013) 27:13391351

embryogenic tissue (ET) initiation (MacKay et al. 2006),

low maturation frequency of the embryogenic tissue
(Montalban et al. 2011a) and poor efficiency in the germination process (Maruyama and Hosoi 2012). Consequently, the number of genotypes that can be candidates for
clonal tests decreases (Davis and Becwar 2007).
To date, as far as we know, Pinus halepensis SE process
has not been studied. Considering the aforementioned
aspects, the general aim of this work was to study the
feasibility of SE in Pinus halepensis using immature
embryos as initial explants.
This general objective included the following tasks: (1)
to determine the optimal explant collection time and
induction medium for initiation of ET and (2) to test the
effects of activated charcoal (AC), sucrose concentration
and nitrogen source in the culture medium on somatic
embryo maturation and conversion into plants.

Materials and methods

Initiation and proliferation
Plant material
One-year-old green female cones (Fig. 1a), enclosing
immature zygotic embryos of Pinus halepensis, were col lava,
lected from open-pollinated trees in Berantevilla (A
Spain).
Intact cones were sprayed with 70 % (v/v) ethanol,
and they were split into quarters and all immature seeds
were removed from the cones. Then, immature seeds
(Fig. 1b) were surface sterilised in 10 % H2O2 (v/v) plus
two drops of Tween 20 for 8 min and then rinsed three
times under sterile distilled H2O in sterile conditions in
the laminar flow unit. Seed coats were removed and
whole megagametophytes containing immature embryos
were excised aseptically and placed horizontally onto the
medium.
Experiment 1
One green cone was sampled weekly, from seven openpollinated families, from the 7th of June to the 16th of
August (eleven collection dates). Then, cones were stored
for a maximum of a week in paper bags at 4 C. These
cones were collected from trees: 1, 2, 3, 4, 5, 6 and 7.
Three basal initiation media (macronutrients, micronutrients and vitamins of these media) were tested: DCR
medium (Gupta and Durzan 1985), EDM medium (Walter
et al. 1998) and LP medium (Quoirin and Lepoivre 1977)
[modified by Aitken-Christie et al. (1988)]. These media
were supplemented with sucrose at 3 % (w/v) and a

Trees (2013) 27:13391351

1341

Fig. 1 Somatic embryogenesis in Pinus halepensis: a green cone

collected on the 12th of July, bar 25 mm; b immature seed from
green cone collected on the 16th of July, bar 7 mm; c initiation and
d proliferation of embryogenic tissue on DCR medium (Gupta and
Durzan 1985), bars 6 mm; e somatic embryo developing on DCR

maturation medium, bar 2 mm; f mature somatic embryo, bar 5 mm;

g somatic embryos germinating on half strength LP medium (Quoirin
and Lepoivre 1977, modified by Aitken-Christie et al. 1988), bar
12 mm; h somatic plant growing in the greenhouse, bar 25 mm

combination of 4.5 lM 2,4-dichlorophenoxyacetic acid

(2,4-D) and 2.7 lM benzyladenine (BA). Before autoclaving, the pH of the media was adjusted to 5.7 and then
3 g L-1 gellan gum (Gelrite) was added. The medium
was autoclaved at 121 C for 20 min. After autoclaving, a
filter-sterilized solution with the pH adjusted to 5.7 containing the ED amino acid mixture (550 mg L-1 L-

glutamine, 525 mg L-1 asparagine, 175 mg L-1 arginine,

19.75 mg L-1 L-citrulline, 19 mg L-1 L-ornithine, 13.75
mg L-1 L-lysine, 10 mg L-1 L-alanine and 8.75 mg L-1 Lproline) was added to the cooled medium prior, and the
media were dispensed into Petri dishes (90 9 20 mm).
Ten megagametophytes per Petri dish, three Petri dishes
per initiation medium, family and collection date were

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Trees (2013) 27:13391351

cultured, resulting in a total number of 6,930 explants. The

Petri dishes were laid out randomly on the shelves of the
growth chamber. Cultures were maintained in the dark at
218 1 C.
After 48 weeks from the beginning of the experiment,
proliferating ET with a size around 35 mm in diameter
was separated from the megagametophytes (Fig. 1c). ET
was sub-cultured to maintenance medium every 2 weeks;
maintenance media had the same composition of the initiation media, but 4 g L-1 of Gelrite. The concentration
of gellan gum in the media was increased to maintain the
spiky morphology of the embryogenic cell lines (ECLs)
(Fig. 1d), according to Breton et al. (2005).
At each collection date, ten megagametophytes per seed
family were destructively sampled, and classification of the
stage of zygotic embryo development was made according
to the eight-stage scale developed by Hargreaves et al.
(2009).
Experiment 2
Cones from two collection dates (28th of July and 2nd of
August) from the families described in Experiment 1 were
collected and stored for a maximum of a week in paper
bags at 4 C.
Ten megagametophytes per Petri dish were cultured. Six
to eight Petri dishes per initiation medium were laid out
randomly on the shelves of the growth chamber. Cultures
were maintained in the dark at 21 1 C.
Twelve initiation media were tested. All media had the
DCR (Gupta and Durzan 1985) basal composition and
differed in:

The amino acid mixture (ED as in experiment 1, or

1 g L-1 casein hydrolysate and 500 mg L-1 Lglutamine).
The growth regulators (DKI mixture: 9 lM 2,4D and
2.7 lM Kinetin; or DBA mixture: 9 lM 2,4D and
2.7 lM BA; or DNB mixture: 4.5 lM 1-naphthaleneacetic acid, 4.5 lM 2,4D and 2.7 lM BA) (Table 1).
The gellan gum concentration: 2 or 3 g L-1 Gelrite.

After 48 weeks, proliferating ET with a size around

35 mm in diameter was separated from the megagametophytes. The ET was sub-cultured to maintenance
medium every 2 weeks; maintenance media had the same
composition of the initiation media but with 4 g L-1 of
gellan gum.
Maturation of ECLs
Plant material
The ECLs from initiation experiment 1 were used for
maturation experiment 1 and ECLs from initiation experiment 2 were used for maturation experiment 2.
Experiment 1
A maturation experiment was designed to assess the effect
of the presence of AC in the ET suspension. The ET was
suspended in liquid growth regulator-free EDM medium
(Walter et al. 1998), supplemented with 2 g L-1 of activated charcoal (AC) (Sigma), or without AC, in 50 mL
centrifuge tubes. Then, ET suspension was vigorously
shaken by hand for a few seconds. Thereafter, a 5 mL
aliquot of the suspension containing 5060 mg fresh
weight (FW) of ET was poured onto a filter paper disc
(Whatman no. 2, 7 cm) in a Buchner funnel. A vacuum
pulse was applied for 10 s, and the filter paper with the
attached ET was transferred to maturation medium. The
maturation medium had the salt formulation of DCR
(Gupta and Durzan 1985) supplemented with 6 % (w/v)
sucrose, 75 lM abscisic acid (ABA), ED amino acid
mixture used for initiation and proliferation of the ET and a
higher concentration of gellan gum (9 g L-1 of Gelrite)(medium C). Sixteen ECLs were tested, three to five
Petri dishes per ECL and treatment were laid out randomly
on the shelves of the growth chamber. Cultures were
maintained in the dark at 21 1 C. After 9 weeks in
maturation medium, the embryos reach the torpedo stage
(Fig. 1e) prior to the cotyledonary one (Fig. 1f) which it is
considered to be the end of maturation phase.
Experiment 2

Table 1 Different plant growth regulator combinations included in

initiation and proliferation media
DBA
(lM)

DKI
(lM)

DNB
(lM)

Benzyladenine

2.7

2.7

Kinetin

2.7

2,4-Dichlorophenoxyacetic
acid

4.5

1-Naphthaleneacetic acid

4.5

123

A maturation experiment was designed to assess the effect

of the composition of maturation medium related to
nitrogen and carbon source on the number of somatic
embryos per gram of ET. The ET was first suspended in
liquid growth regulators-free EDM medium (Walter et al.
1998) and a 5 mL aliquot containing 7080 mg of suspended ET (FW) was filtered and transferred to maturation
medium as described above. Four maturation media were
tested; one of them was described in experiment 1 (medium

Trees (2013) 27:13391351

1343

number for experiment 1 and with h followed by a number

for experiment 2.

C). All maturation media had the salt formulation of DCR

(Gupta and Durzan 1985), 75 lM ABA and 9 g L-1 of
Gelrite. These media were supplemented with sucrose at
4.5 % (maturation media A and B) or 6 % (maturation
media C and D), and ED amino acid mixture used for
initiation and proliferation of the ET (maturation media A
and C) or the same amino acid mixture but with a higher
concentration of glutamine (1,650 mg L-1 instead of the
550 mg L-1) (maturation media B and D) (Table 2). Three
ECLs were tested; three Petri dishes per ECL and treatment
were laid out randomly on the shelves of the growth
chamber. Cultures were maintained in the dark at
21 1 C.

Maturation
After 18 weeks from the beginning of the experiments, the
number of mature somatic embryos per Petri dish was
recorded and the number of mature somatic embryos per
gram was calculated.
Germination
After 18 weeks on germination medium the number of
germinated somatic embryos per gram of ET and the
overall germinated somatic embryos related to the total
number of somatic embryos introduced (conversion, %)
was calculated.
The results for all the experiments carried out were
analysed by ANOVA, and significant differences between
means were determined by the Tukey post hoc test at a
significance level of p \ 0.05.

Germination
Mature somatic embryos from maturation experiments
(Fig. 1f) were collected and cultured on half strength LP
[(Quoirin and Lepoivre 1977) modified by Aitken-Christie
et al. (1988)] supplemented with 2 g L-1 AC and 10 g L-1
Difco agar granulated. Ten to twenty mature somatic
embryos per Petri dish were cultured.
The plants (Fig. 1g) were sub-cultured once onto fresh
medium of the same composition every 6 weeks. Cultures
were maintained at 21 1 C under a 16-h photoperiod at
120 lmol m-2s-1 provided by cool white fluorescent tubes
(TLD 58 W/33; Philips, France).
After 1824 weeks on germination medium, the plants
were transferred to sterile peat: perlite (7:3, v/v) and
acclimatized in a greenhouse under controlled temperatures
at 21 2 C and progressively decreasing humidity from
90 to 60 % (Fig. 1h).

Results
Initiation and proliferation
Experiment 1
From the 2,310 megagametophytes cultured on EDM
medium, only one initiation of ET was observed and it did
not continue to proliferate. On LP medium, 25 initiations of
ET from the 2,310 megagametophytes cultured were
recorded; 52 % of these initiations were obtained from
families 4 and 5 at collection dates 10 and 11. From the 25
initiations observed, only three of them led to ECLs (data
not shown).
DCR medium gave the best results. The initiation of ET
on DCR medium was significantly affected by the seed
family and the collection date tested (Table 3). There was
also a significant (p \ 0.01) interaction among the mother
trees and the weeks studied (Table 3). Family 4 produced a
significantly (p B 0.05) higher initiation percentage
(25.6 %) than family 1 (8.3 %) (Table 4). The rest of the

Data collection and statistical analysis

Initiation and proliferation experiments
After 810 weeks from the beginning of the experiments,
the number of proliferating ET calli with a size around
35 mm in diameter were recorded and initiation percentage per Petri dish was calculated. Following three
subculture periods, actively growing ETs were recorded as
ECLs and proliferation percentage per Petri dish was calculated. Each ECL was named with letter H followed by a
Table 2 Description of the
different maturation media
tested, giving amino acid
composition and sucrose
percentage

Sucrose (%)

Amino acid mixture

Maturation medium A

4.5

ED amino acid mixture (550 mg L-1 glutamine)

Maturation medium B

4.5

ED mixture (1,650 mg L-1 glutamine)

Maturation medium C
Maturation medium D

6
6

ED amino acid mixture (550 mg L-1 glutamine)

ED mixture (1,650 mg L-1 glutamine)

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Table 3 ANOVA for embryogenic tissue initiation and proliferation (%) in Pinus halepensis megagametophytes from seven open-pollinated
trees cultured on DCR medium (Gupta and Durzan 1985) at six collection dates
Source

df

ET Initiation (%)
Mean square

ET Proliferation (%)
F test

p value

Mean square

F test

p value
\0.05

Seed family (S)

724.07

3.02

\0.01

140.21

2.90

Collection date (C)

1,441.43

6.01

\0.001

94.13

1.94

n.s.

30
84

715.50
239.68

2.99

\0.01

72.28
239.68

1.49

n.s.

S9C
Error
n.s. non-significant

families presented initiation percentage means ranging

from 16.1 to 23.9 and did not show significant differences
either with family 4 or with family 1 (Table 4).
When collection dates were considered, the megagametophytes cultured on initiation medium from the 7th of
June to the 5th of July (collection dates 1, 2, 3, 4 and 5) did not
initiate ET (data not shown). The best results for initiation of
ET were obtained between collection dates 9 and 10 (27.1
and 28.1 %, respectively) (Table 4); these collection dates
correspond to zygotic embryo developmental stages
between 3 and 4 (early bullet stage embryos). The results
obtained at collection dates 9 and 10 were significantly
(p B 0.05) higher than initiation percentage at collection
date 8 (6.7 %) (Table 4). The other collection dates studied
showed initiation percentages ranging from 14.3 to 19.5 and
did not show significant differences either with collection
dates 9 and 10 or with collection date 8 (Table 4).

In summary, the highest initiation percentage (70 %)

was obtained in family 4 in collection date 10. Taking
into account the interaction among seed families and
collection dates, six from the seven seed families studied
achieved their peak initiation percentages between collection dates 9 and 11 (Table 4). Family 7 showed the
highest initiation of ET at collection date 6, whereas
family 2 had equal initiation percentages at collection
dates 6 and 9 (Table 4).
Regarding ET proliferation, this was significantly
(p \ 0.05) affected by the seed family studied (Table 3). In
this sense, family 3 had significantly (p \ 0.05) better
values (7.2 %) than families 1, 2 and 5 with proliferation
percentages lower than 1.2 % (Fig. 2). Families 4, 6 and 7
whose proliferation percentages ranged from 4.4 to 6.7 %
did not show significant differences with the other families
tested (Fig. 2).

Table 4 Embryogenic tissue (ET) initiation (%) in Pinus halepensis in seven open-pollinated trees cultured on DCR medium (Gupta and Durzan
1985) at six collection dates, different letters show significant differences at p \ 0.01 by Tukeys post hoc test (M SE)
Seed
family

ET initiation (%)
Collection date
6th
12 July

7th
19 July

6.7 6.7cd

10.0 5.8cd

30.0 0.0

abcd

16.7 8.8

bcd

0.0 0.0d

0.0 0.0

0.0 0.0

6
7

8th
26 July

cd

6.7 6.7

26.7 6.7

abcd

23.3 3.3abcd
10.0 10

cd

cd

6.7 3.3

46.7 24.0

abcd

ab

16.7 3.3

bcd

14.3 2.5

ab

9th
2 August

0.0 0.0d

10th
9 August

6.7 6.7cd

10.0 5.8cd

30.0 5.8

abcd

3.3 3.3

26.7 8.8

abcd

20.0 11.6abcd

26.7 8.8abcd

10.0 5.8

cd

cd

0.0 0.0

16.7 16.7

0.0 0.0

13.3 3.3

bcd

11th
16 August

bcd

63.3 8.8

ab

20.0 0.0

abcd
a

cd

6.7 6.7

53.3 8.8

abc

70.0 0.0a

Mean

16.7 8.8bcd
13.3 6.7

16.1 3.1ab

16.7 3.3

bcd

23.9 4.4ab

13.3 8.8bcd

16.7 12.0

bcd

26.7 21.9

abcd

13.3 8.8

bcd

28.1 6.1

8.3 2.5b

bcd

25.6 5.8a

30.0 15.3

abcd

12.2 4.6ab

33.3 17.6

abcd

21.7 6.9ab

13.3 3.3

bcd

19.5 3.8

ab

Mean

14.3 4.9

6.7 2.3

27.1 4.7

Embryo
stage

1.5

(Proembryonary
stage)

(Early cleavage polyembryonary stage)

(Cleavage polyembryonary
stage)

(Early bullet
stage)

(Bullet stage)

(Late bullet
stage)

20.6 4.7ab

In the last row, the embryo stage is shown. Means followed by the same letter (a, b, c or d) are not significantly different at p \ 0.05 Tukeys post
hoc test

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Maturation and germination

ET Proliferation (%)

25
20

Experiment 1

15
a

ab

10

ab

ab

5
b

0
3

Seed family

Fig. 2 Embryogenic tissue (ET) proliferation (%) in megagametophytes from seven seed families of Pinus halepensis cultured on DCR
medium (Gupta and Durzan 1985) (M SE). Different letters show
significant differences at p \ 0.05 by Tukeys post hoc test

Experiment 2
In this experiment twelve media were tested. These media
differed in the nitrogen source, the plant growth regulator
combinations or the gellan gum concentration. The
ANOVA showed that only the nitrogen source used had a
significant effect on ET initiation and no interaction was
observed (Table 5). Thus, the media containing ED amino
acid mixture presented an initiation percentage of 36.4 %,
whereas the media supplemented with 1 g L-1 casein
hydrolysate and 500 mg L-1 L-glutamine had an initiation
percentage of 15.4 % (data not shown).
When analysing the proliferation stage, it was observed
that this was only significantly (p \ 0.05) influenced by the
growth regulators (Table 5). At this stage, we also
observed no interactions between the factors studied.
Namely, the combinations of growth regulators DKI and
DNB gave significantly (p \ 0.05) better results (12 and
11.5 %, respectively) than DBA (3.3 %) (Fig. 3).

Table 5 ANOVA for embryogenic tissue (ET) initiation and proliferation (%) in Pinus halepensis megagametophytes cultured on DCR
medium (Gupta and Durzan 1985) supplemented with different amino
acid mixtures [(A), ED or 1 g L-1 casein hydrolysate and
Source

df

From the sixteen ECLs tested, thirteen ECLs produced

mature somatic embryos. The ANOVA for these thirteen
ECLs showed that the ECL (p \ 0.001) and the interaction
among ECL and AC (p \ 0.05) had a significant effect on
the maturation process (Table 6). Notably, H49 and H150
only produced somatic embryos when the suspension used
for maturation had AC whereas H58 developed somatic
embryos solely when the suspension lacked AC (Fig. 4).
The ECL H29 was more productive (716 somatic
embryos per gram of ET) than the rest of the ECLs
assayed. Six ECLs produced between 108 and 350 somatic
embryos per gram of ET, whereas six ECLs produced less
than 68 somatic embryos per gram of ET (Fig. 4).
When considering the interaction between the ECL and
presence or absence of AC in the suspension used for
maturation, in five of the thirteen ECLs tested, the presence
of AC was beneficial (Fig. 4). On the contrary, in eight
ECLs the absence of AC in the suspension led to a higher
production of somatic embryos (Fig. 4).
The number of germinated somatic embryos was significantly (p \ 0.001) affected by the ECL tested (Table 6)
and there was no interaction among the ECL and the
presence or absence of AC in the suspension used for
maturation. Three of the thirteen ECLs tested yielded more
than 100 germinated somatic embryos per gram of ET.
Only two of the lines tested produced less than 10 embryos
per gram of ET (Table 7).
Considering the conversion rate, the overall germination
percentage in this experiment was 60 % (data not shown).
The plants were successfully acclimatized in plastic

500 mg L-1 L-glutamine], plant growth regulator combinations

[(H), DBA, DKI and DNB] or gellan gum concentrations [(G),
2 g L-1 or 3 g L-1]

ET initiation (%)

ET proliferation (%)

Mean square

F test

p value

Mean square

F test

p value

Amino acid mixture (A)

8,294.81

18.22

\0.001

443.41

3.12

n.s.

Growth regulators (H)

132.90

0.29

n.s.

594.14

4.19

\0.05
n.s.

Gellan gum (G)

1.56

0.003

n.s.

208.10

1.47

A9H

182.13

0.40

n.s.

133.98

0.94

n.s.

A9G
H9G

1
2

380.52
143.81

0.83
0.31

n.s.
n.s.

0.54
206.37

0.004
1.45

n.s.
n.s.

15.29

0.03

n.s.

119.45

0.84

n.s.

64

457.73

A9H9G
Error

141.96

n.s. non-significant

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ET Proliferation (%)

25
20
a

15
10
b

5
0
DKI

DBA

DNB

Plant growth regulator combinations

Fig. 3 Embryogenic tissue (ET) proliferation (%) in Pinus halepensis

megagametophytes cultured on DCR medium (Gupta and Durzan
1985) supplemented with plant growth regulators [(9 lM 2,4-D and
2.7 lM Kinetin (DKI); 9 lM 2,4-D and 2.7 lM BA (DBA); or
4.5 lM NAA and 4.5 lM 2,4-D and 2.7 lM BA (DNB)] (M SE).
Different letters show significant differences at p \ 0.05 by Tukeys
post hoc test

propagation trays, and their growth is currently being

monitored in the greenhouse.
Experiment 2
The ECL, the maturation medium (A, B, C or D) and the
interaction between them had a significant (p \ 0.001)
effect on the number of mature somatic embryos obtained
per gram of ET (Table 8). The ECL h42 yielded a significantly (p \ 0.05) higher number of somatic embryos (195
somatic embryos per gram of ET) than h19 and h41 (57 and
114 somatic embryos per gram of ET, respectively)
(Table 9). The maturation medium C led to a significantly
(p B 0.05) higher production of somatic embryos (243
somatic embryos per gram of ET) than the other media
tested (from 70.5 to 97.2 somatic embryos per gram of ET)
(Table 9).
The interaction among ECL and maturation medium
showed that ET from h41 and h42 performed better when
cultured on maturation medium C (247 and 435 somatic
embryos per gram of ET), whereas h19 produced its higher

number of somatic embryos (81 somatic embryos per gram

of ET) on maturation medium B (a medium with lower
concentration of sucrose and a higher concentration of
glutamine) (Table 9).
The results regarding germination of the somatic
embryos followed the same trend as for the maturation
process (Table 8). Thus the ECL, the maturation medium
and the interaction between them had a significant
(p \ 0.001) effect on the number of germinated somatic
embryos obtained per gram of ET (Table 8). Again, h42
produced a significantly (p \ 0.05) higher number of germinated somatic embryos (172) per gram of ET than the
other ECLs tested (Table 10). In this sense ET on maturation medium C also yielded a significantly (p \ 0.05)
higher number of germinated somatic embryos (207) per
gram of ET than the other maturation media tested
(Table 10).
The interaction among ECL and maturation medium
also showed that the highest number of germinated somatic
embryos was obtained on medium C for h41 and h42,
whereas for h19 the best results were found on medium B
(Table 10). The overall conversion percentage in this
experiment was 80 % (data not shown). The plantlets were
successfully acclimatized in plastic propagation trays, and
their growth is currently being monitored in the
greenhouse.

Discussion
In a first experiment the effect of culture medium, collection date and seed family on ET initiation and proliferation
in Pinus halepensis were analysed. Our results show that
these SE stages were strongly affected by culture medium
with the best results obtained after culture on DCR (Gupta
and Durzan 1985) medium. This medium was also found to
be the best for organogenesis in this species (Lambardi
et al. 1993) and for organogenesis and SE in other Pinus

Table 6 ANOVA for the number of mature somatic embryos and germinated somatic embryos per gram (FW) of embryogenic tissue (ET) in
Pinus halepensis
Source

df

Somatic embryos per gram of ET

Germinated somatic embryos per gram of ET

Mean square

F test

p value

Mean square

F test

p value

\0.001

\0.001

ECL

12

263,388.29

23.36

61,330.15

13.49

AC

10,520.27

0.93

n.s.

3,617.36

0.80

n.s.

12

21,929.27

1.94

\0.05

3,226.48

0.71

n.s.

100

11,274.42

ECL 9 AC
Error

4,547.41

The embryogenic tissue from thirteen embryogenic cell lines (ECLs) was suspended in liquid EDM medium, supplemented with 2 g L-1 of
activated charcoal (AC) or without AC. Somatic embryos were germinated on half strength LP (Quoirin and Lepoivre 1977, modified by AitkenChristie et al. 1988) supplemented with 2 g L-1 AC
n.s. non-significant

123

1347

1000
800
600
400
200

H29

H153

H217

H30

H239

H195

H47

H45

H32

H60

H150

H58

0
H49

N of mature somatic embryos per gram of

embryogenic tissue (FW)

Trees (2013) 27:13391351

Embryogenic cell lines

Fig. 4 Mature somatic embryos per gram (FW) of embryogenic

tissue (ET) from thirteen embryogenic cell lines (ECLs) suspended in
liquid EDM medium, with 2 g L-1 of activated charcoal (AC) (black
squares linked by a dotted line), or without AC (white squares linked
by a solid line) (M SE)
Table 7 Germinated somatic embryos per gram (FW) of embryogenic tissue (ET) in Pinus halepensis (M SE)
ECL

Germinated somatic embryos

per gram (FW) of ET

H29
H30
H32
H45
H47
H49
H58
H60
H150
H153
H195
H217
H239

270.0
30.0
32.3
19.5
30.2
4.0
10.0
11.0
0.0
101.8
58.2
171.0
42.6

52.8a
10.9c
11.7c
9.0c
15.5c
4.0c
8.0d
6.2c
0.0c
20.6bc
15.8c
32.9ab
11.0c

The somatic embryos were germinated on half strength LP (Quoirin

and Lepoivre 1977, modified by Aitken-Christie et al. 1988) supplemented with 2 g L-1 of activated charcoal
Means followed by the same letter (a, b, c or d) are not significantly
different at p \ 0.05 Tukeys post hoc test

spp. (Miguel et al. 2004; De Diego et al. 2008, 2010;

Aronen et al. 2009). On DCR medium (Gupta and Durzan
1985), ET initiation rates could be significantly affected by
the collection date (Table 4), in agreement with reports on
several Pinus species (Klimaszewska et al. 2001; Hargreaves et al. 2009; Montalban et al. 2012a).
SE in Pinus halepensis was initiated in all of the seed
families tested and only differences among two seed families were observed, with initiation percentages ranging
from 8.3 to 25.6 % (Table 4). These ET initiation rates are
consistent with reported results in P. taeda (Pullman et al.
2003). Proliferation of ET ranged depending on the family,
from 0.6 to 7.2 % (Fig. 2), in accordance with results in
other Pinus species (Maruyama et al. 2007). At the proliferation stage no significant effect of the collection date
was observed (Table 3) which accords with Hargreaves
et al. (2011); this is contrary to the results reported by
Yildirim et al. (2006) in P. brutia or Montalban et al.
(2012a) in P. radiata SE. In a second ET initiation and
proliferation experiment, the effect of different nitrogen
sources, growth regulator combinations and gellan gum
concentrations on the culture medium was assessed. Our
results showed that ET initiation was significantly affected
by the nitrogen source, whereas proliferation was significantly affected by the combination of growth regulators
supplemented in the culture medium (Table 5). Initiation
was better when the culture medium was supplemented
with ED amino acid mixture. Although in previous studies
in P. radiata SE we reported that the amino acid mixture
1 g L-1 casein hydrolysate plus 500 mg L-1 L-glutamine
yielded better proliferation rates, we also found that ET
cultured in the presence of ED amino acid mixture performed better in the long term (Montalban et al. 2012a).
The rates of ET proliferation in this second experiment
were significantly better when the culture medium was
supplemented with DKI or DNB plant growth regulator
combinations. The three plant growth regulator combinations used had 2.7 lM cytokinin and 9 lM auxin. In some
cases, the addition of cytokinins as the sole source of plant
growth regulators is sufficient to induce and maintain SE

Table 8 ANOVA for the number of mature somatic embryos and the number of germinated somatic embryos per gram (FW) of embryogenic
tissue (ET) in Pinus halepensis
Source

df

Somatic embryos per gram of ET

Germinated somatic embryos per gram of ET

Mean square

F test

p value

Mean square

F test

p value

Embryogenic cell line (ECL)

58,053.39

15.71

\0.001

50,344.79

17.17

\0.001

Maturation medium (M)

60,029.64

19.25

\0.001

46,334.89

15.80

\0.001

6
24

22,196.51
3,694.79

6.01

\0.001

19,909.98
2,932.41

6.79

\0.001

ECL 9 M
Error

The ET was cultured on different maturation media (A, B, C and D). Somatic embryos were germinated on half strength LP (Quoirin and
Lepoivre 1977, modified by Aitken-Christie et al. 1988) supplemented with 2 gL-1 of activated charcoal

123

1348

Trees (2013) 27:13391351

Table 9 Mature somatic embryos per gram (FW) of embryogenic tissue (ET) in Pinus halepensis (M SE)
ECL

Somatic embryos per gram of ET

Maturation media

Mean

Low sucrose

High sucrose

Low glutamine
A

High glutamine
B

Low glutamine
C

h19

28.6 14.3c

81.0 26.5bc

47.6 47.6c

h41

89.7 7.4

bc

h42

115.6 27bc

Mean

78.0 15.8

68.4 23.8

142.2 49.5bc
b

97.2 21.0

High glutamine
D
71.4 16.5bc
c

247.9 52.5

51.3 31.2

435.6 54.1a

88.9 17.8bc

243.7 61.6

70.5 14.4

57.1 14.0b
114.3 27.9b
195.6 45.5a

The ET was cultured on different maturation media (A, B, C and D). Different letters show significant differences at p \ 0.001 by Tukeys post
hoc test. In the last row and the last column appear the mean for the number of somatic embryos (%) on the ECLs and maturation media tested,
respectively (M SE)
Means followed by the same letter (a, b, c or d) are not significantly different at p \ 0.05 Tukeys post hoc test

Table 10 Germinated somatic embryos per gram (FW) of embryogenic tissue (ET) in Pinus halepensis (M SE)
ECL

Germinated somatic embryos per gram of ET

Maturation media
Low sucrose
Low glutamine
A

High sucrose
High glutamine
B

Low glutamine
C
33.3 33.3bc

h19

28.6 14.3c

66.7 26.5bc

h41

bc

64.1 19.6

bc

115.6 49.5

bc

h42
Mean

72.7 8.6

bc

97.8 19.4

66.3 12.5

Mean

82.1 19.1

188.0 40.8

400.0 61.1

207.1 58.0

High glutamine
D
52.4 17.2bc
21.4 11.3

75.6 24.8

bc

49.8 12.2

45.2 11.2b
86.5 21.2b
172.2 43.8a

The ET was cultured on different maturation media (A, B, C and D). Somatic embryos were germinated on half strength LP (Quoirin and
Lepoivre 1977, modified by Aitken-Christie et al. 1988) supplemented with 2 g L-1 of activated charcoal (AC). Different letters show significant
differences at p \ 0.001 by Tukeys post hoc test. In the last row and the last column appear the mean for the number of somatic embryos (%) on
the ECLs and maturation media tested, respectively (M SE)
Means followed by the same letter (a, b, c or d) are not significantly different at p \ 0.05 Tukeys post hoc test

(Krajnakova et al. 2008), but most of the protocols used

auxins (alone or in combination with cytokinins) for SE
induction (Gaj 2004; Klimaszewska et al. 2007). Although
many authors use for proliferation the same or slightly
modified initiation medium, the results obtained for initiation and proliferation suggest that a multi-step protocol
would be better. Montalban et al. (2012b) described a
protocol to induce organogenesis in P. radiata buds from
adult trees and containing two steps, one in presence of
high cytokinin concentration and another in absence of
plant growth regulators. Furthermore, Fernandez-Guijarro
et al. (1995) in Quercus suber SE reduced the high concentrations of BAP and NAA present in a first step of the
process to lower levels in a second one.
The ECLs obtained in the aforementioned experiments
were subjected to maturation. In maturation experiment
1, where the effect of AC in the ET suspension was

123

assessed, over 80 % of the lines tested produced somatic

embryos. In this case, our results showed that the ECL
and the interaction among ECL and AC had a significant
effect on the number of somatic embryos produced
(Table 6). The mean numbers of somatic embryos per
gram of ET ranged from 7 to 716 (Fig. 4); these results
are consistent with others obtained in P. radiata
(Montalban et al. 2010) or in P. pinaster (Lelu-Walter
et al. 2006). When the interaction between ECLs and AC
was considered, a clear trend was not found (Fig. 4).
Similar results were obtained in P. sylvestris SE (LeluWalter et al. 2008) where the effect of AC depended on
the line tested. AC plays an essential role in micropropagation of conifers for its capacity of adsorbing
phenolics and residual plant growth regulators (Thomas
2008; De Diego et al. 2010), but its effect on maturation
depends also on the species tested, being beneficial in

Trees (2013) 27:13391351

some of them and detrimental in others (Montalban et al.

2010).
In a second maturation experiment, the effect of sucrose
concentrations and different nitrogen sources (with high or
standard glutamine concentration) on the maturation
medium was assessed. The ANOVA for the number of
somatic embryos per gram of ET showed a significant
effect of both factors analysed (ECL and maturation
medium) and a significant effect of the interaction between
factors (Table 8). Previous research with Tamarillo has
shown that the manipulation of sucrose concentration in the
development medium increased the number of morphologically normal somatic embryos (Correia et al. 2012).
Furthermore, several authors (Kumar and Thomas 2012;
Rode et al. 2012) demonstrated that the addition of higher
concentrations of sucrose with and without higher ABA
concentrations significantly increased the embryogenic
response. The effect of sucrose concentration has been also
widely studied in different Pinus species and, as discussed
by Yildirim et al. (2006), the suitability of a higher or a
lower sucrose concentration for maturation depends on the
species studied. Previous work carried out with radiata pine
in our laboratory showed a significant increase of the
number of somatic embryos per gram of ET by increasing
the sucrose concentration (from 30 to 60 g L-1) (Montalban et al. 2010). However, as Montalban et al. (2010)
demonstrated and Percy et al. (2001) discussed, variation
among ECLs for maturation yields is an attribute common
to several conifer species. Some studies have shown the
importance of nitrogen source compounds for the proliferation and maturation of somatic embryos in different
species (Perez-Rodrguez et al. 2006). Gerdakanenh et al.
(2011) postulated that stimulation of embryogenesis and
embryo development was strictly dependent on the type
and concentration of amino acid in the medium. Mihaljevic
et al. (2011) have shown in Cucurbita pepo L. that later
stage embryos developed only after a re-supply of nitrogen
in the form of nitrate or L-glutamine. In pine SE, it has been
suggested that the carbohydrate to nitrogen ratio of the
culture medium may represent a key factor responsible for
the expression of certain glutamine synthetase-related and
photosynthesis-related genes (Perez-Rodrguez et al. 2006).
In our experiment, in the most productive lines, the maturation medium with a low concentration of glutamine and
a high concentration of sucrose led to the highest number
of somatic embryos per gram of ET, whereas in the less
productive line, the best results were obtained after culture
of ET on a maturation medium with a high concentration of
glutamine and a low concentration of sucrose (Table 9).
Ramarosandratana et al. (2001) also pointed out that
somatic embryo maturation in maritime pine was highly
variable and depended both on the ECL and the maturation
medium. Moreover, Divakaran and Nair (2011) observed

1349

the different effect of increasing L-glutamine concentration

in the number of germinated embryos in Musa acuminata.
The somatic embryos obtained in both maturation
experiments were successfully germinated on half strength
LP (Quoirin and Lepoivre 1977, modified by AitkenChristie et al. (1988)), with conversion percentages ranging
from 60 to 80 %, which is in accordance with the results
reported by Choudhury et al. (2008) in P. kesiya and Percy
et al. (2000) in P. monticola. Although germination has
been accomplished in other Pinus species with a postmaturation treatment (Maruyama and Hosoi 2012) or on
media without AC (Klimaszewska et al. 2001), this germination procedure and medium has been proven to be
suitable for micropropagation in species such as P. radiata
(Montalban et al. 2010; Montalban et al. 2011b).
To the best of our knowledge, this is the first report on
P. halepensis SE for which we have developed a simple
protocol that can be an efficient procedure for large-scale
somatic embryo production. The authors consider that this
protocol will be a very useful tool for the propagation of
selected families/crosses and for genetic engineering of this
species.
Acknowledgments We thank Charlie B. Low for his statistical
advice. This research was funded by Ministerio de Ciencia y TecnologaSpain (AGL2005-08214-CO2-02) and Departamento de
Agricultura y Pesca-Gobierno Vasco (VEC2004021 and
VED2007014), who granted I. A. M. with a PhD scholarship.

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