34 somatic embryogenesis in pinus halepensis mill an important ecological species from the...

8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest

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O R I G I N A L P A P E R

Somatic embryogenesis in Pinus halepensis Mill.: an importantecological species from the Mediterranean forest

I. A. Montalbán • A. Setién-Olarra •

C. L. Hargreaves • P. Moncaleán

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 forenvironmental 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 regula-

tors), collection date and seed family on embryogenic tis-

sue 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 col-

lection dates. Furthermore, the embryogenic tissue initia-

tion 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 effi-

cient procedure for large-scale somatic embryo production.

Keywords Aleppo pine Embryogenic tissue

Germination

Initiation

Maturation

Propagation

Abbreviations

A Maturation medium, DCR basal mediumsupplemented 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

Communicated by K. Klimaszewska.

I. A. Montalbán A. Setién-Olarra P. Moncaleán (&)

Centro de Arkaute, Neiker-Tecnalia, Apdo. 46,

01080 Vitoria-Gasteiz, Spain

e-mail: [email protected]

C. L. Hargreaves

Scion, Private Bag 3020, Rotorua 3046, New Zealand

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DOI 10.1007/s00468-013-0882-0


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Introduction

Pinus halepensis Mill., commonly named Aleppo pine, is a

species native to the Mediterranean region and is wide-

spread from Spain to Algeria (Botella et al. 2010). Tem-

perature and precipitation requirements generally confine

its distribution to sub-humid areas of the Mediterranean. Its

main area of distribution is Southern Europe (Spain, Italyand 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 mar-

ginal and submarginal areas because it is one of the most

drought resistant pine species (Klein et al. 2011). More-

over, in marine stands, P. halepensis forests are importantboth 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 (Abelló 1998).

In vitro vegetative propagation from physiologically

juvenile tissue has been successful in a number of conifer

species (Moncaleán et al. 2005). This phenomenon has led

many organizations to focus on production of elite families

through juvenile tissue propagation in the short-term, eitherfor 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) tradi-

tional breeding strategies combined with in vitro vegetative

propagation have shown advantages. These include addi-

tional 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, propa-

gation via somatic embryogenesis (SE) from immature

zygotic embryos is an effective method of propagation

when combined with other technologies, such as cryopre-

serving 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 (Montalbán et al. 2010) as well as to use

the somatic embryos for organogenesis procedures and thus

provide further amplification (Montalbán et al. 2011a).

However, this technology has a number of bottlenecks in

the Pinus genus, namely, a narrow competence window for

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

low maturation frequency of the embryogenic tissue

(Montalbán et al. 2011a) and poor efficiency in the ger-

mination process (Maruyama and Hosoi 2012). Conse-

quently, 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 aforementionedaspects, 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-

lected from open-pollinated trees in Berantevilla (Álava,

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 open-

pollinated 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, micronu-

trients 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

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combination of 4.5 lM 2,4-dichlorophenoxyacetic acid

(2,4-D) and 2.7 lM benzyladenine (BA). Before auto-

claving, 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 con-

taining 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 L-

proline) 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

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 andDurzan 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

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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 4–8 weeks from the beginning of the experiment,

proliferating ET with a size around 3–5 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 ini-

tiation 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 L-

glutamine).

• 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-naphthalene-

acetic 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 4–8 weeks, proliferating ET with a size around

3–5 mm in diameter was separated from the megaga-

metophytes. 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 exper-

iment 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 acti-

vated 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 50–60 mg fresh

weight (FW) of ET was poured onto a filter paper disc

(Whatman no. 2, 7 cm) in a Büchner 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 Gel-

rite)(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

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 70–80 mg of sus-

pended 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

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

9 9 4.5

1-Naphthaleneacetic acid – – 4.5

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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 the550 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.

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 18–24 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).

Data collection and statistical analysis

Initiation and proliferation experiments

After 8–10 weeks from the beginning of the experiments,

the number of proliferating ET calli with a size around

3–5 mm in diameter were recorded and initiation per-

centage per Petri dish was calculated. Following three

subculture periods, actively growing ETs were recorded as

ECLs and proliferation percentage per Petri dish was cal-

culated. Each ECL was named with letter H followed by a

number for experiment 1 and with h followed by a number

for experiment 2.

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 pergram 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 asignificance level of p \ 0.05.

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

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 6 ED amino acid mixture (550 mg L-1

glutamine)

Maturation medium D 6 ED mixture (1,650 mg L-1

glutamine)

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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 megaga-

metophytes cultured on initiation medium from the 7th of

Junetothe5thofJuly(collectiondates1,2,3,4and5)didnot

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 col-

lection 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 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 (%) ET Proliferation (%)

Mean square F test p value Mean square F test p value

Seed family (S) 6 724.07 3.02 \0.01 140.21 2.90 \0.05

Collection date (C) 5 1,441.43 6.01 \0.001 94.13 1.94 n.s.

S 9 C 30 715.50 2.99 \0.01 72.28 1.49 n.s.

Error 84 239.68 239.68

n.s. non-significant

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 Tukey’s post hoc test (M ± SE)

Seed

family

ET initiation (%)

Collection date

6th 7th 8th 9th 10th 11th Mean

12 July 19 July 26 July 2 August 9 August 16 August

1 6.7 ± 6.7cd 10.0 ± 5.8cd 0.0 ± 0.0d 6.7 ± 6.7cd 10.0 ± 5.8cd 16.7 ± 8.8bcd 8.3 ± 2.5b

2 30.0 ± 0.0abcd

6.7 ± 6.7cd

10.0 ± 5.8cd

30.0 ± 5.8abcd

6.7 ± 6.7cd

13.3 ± 6.7bcd

16.1 ± 3.1ab

3 16.7 ± 8.8bcd 26.7 ± 6.7abcd 3.3 ± 3.3cd 26.7 ± 8.8abcd 53.3 ± 8.8abc 16.7 ± 3.3bcd 23.9 ± 4.4ab

4 0.0 ± 0.0d 23.3 ± 3.3abcd 20.0 ± 11.6abcd 26.7 ± 8.8abcd 70.0 ± 0.0a 13.3 ± 8.8bcd 25.6 ± 5.8a

5 0.0 ± 0.0d 10.0 ± 10cd 0.0 ± 0.0d 16.7 ± 16.7bcd 16.7 ± 12.0bcd 30.0 ± 15.3abcd 12.2 ± 4.6ab

6 0.0 ± 0.0d 6.7 ± 3.3cd 0.0 ± 0.0d 63.3 ± 8.8ab 26.7 ± 21.9abcd 33.3 ± 17.6abcd 21.7 ± 6.9ab

7 46.7 ± 24.0abcd 16.7 ± 3.3bcd 13.3 ± 3.3bcd 20.0 ± 0.0abcd 13.3 ± 8.8bcd 13.3 ± 3.3bcd 20.6 ± 4.7ab

Mean 14.3 ± 4.9ab

14.3 ± 2.5ab

6.7 ± 2.3b

27.1 ± 4.7a

28.1 ± 6.1a

19.5 ± 3.8ab

Embryo

stage

1 1.5 2 3 4 5

(Pro-

embryonary

stage)

(Early cleavage poly-

embryonary stage)

(Cleavage poly-

embryonary

stage)

(Early bullet

stage)

(Bullet stage) (Late bullet

stage)

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 Tukey’s post

hoc test

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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. TheANOVA 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 andDNB gave significantly ( p\ 0.05) better results (12 and

11.5 %, respectively) than DBA (3.3 %) (Fig. 3).

Maturation and germination

Experiment 1

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 onthe 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 presenceof 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 sig-

nificantly ( 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

0

5

10

15

20

25

1 2 3 4 5 6 7

Seed family

E T P r o l i f e r a t i o n ( % )

b b

a

ab

b

ab

ab

Fig. 2 Embryogenic tissue (ET) proliferation (%) in megagameto-

phytes 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 Tukey’s post hoc test

Table 5 ANOVA for embryogenic tissue (ET) initiation and prolif-

eration (%) 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

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

]

Source df ET initiation (%) ET proliferation (%)


Amino acid mixture (A) 1 8,294.81 18.22 \0.001 443.41 3.12 n.s.

Growth regulators (H) 2 132.90 0.29 n.s. 594.14 4.19 \0.05

Gellan gum (G) 1 1.56 0.003 n.s. 208.10 1.47 n.s.

A 9 H 2 182.13 0.40 n.s. 133.98 0.94 n.s.

A 9 G 1 380.52 0.83 n.s. 0.54 0.004 n.s.

H 9 G 2 143.81 0.31 n.s. 206.37 1.45 n.s.

A 9 H 9 G 2 15.29 0.03 n.s. 119.45 0.84 n.s.

Error 64 457.73 141.96


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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 signif-

icantly ( 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 ger-

minated somatic embryos (172) per gram of ET than the

other ECLs tested (Table 10). In this sense ET on matu-

ration 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, collec-

tion 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

0

5

10

15

20

25

DKI DBA DNB

Plant growth regulator combinations

E T P r o l i f e r a t i o n ( % )

aa

b

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 Tukey’s

post hoc test

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


ECL 12 263,388.29 23.36 \0.001 61,330.15 13.49 \0.001

AC 1 10,520.27 0.93 n.s. 3,617.36 0.80 n.s.

ECL 9 AC 12 21,929.27 1.94 \0.05 3,226.48 0.71 n.s.

Error 100 11,274.42 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 Aitken-

Christie et al. 1988) supplemented with 2 g L-1

AC


1346 Trees (2013) 27:1339–1351

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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; Har-

greaves et al. 2009; Montalbán et al. 2012a).

SE in Pinus halepensis was initiated in all of the seed

families tested and only differences among two seed fam-ilies 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 pro-

liferation 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 Montalbán et al.

(2012a) in P. radiata SE. In a second ET initiation and

proliferation experiment, the effect of different nitrogensources, 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 signifi-

cantly 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 per-

formed better in the long term (Montalbán 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 combina-

tions 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 halepensisSource df Somatic embryos per gram of ET Germinated somatic embryos per gram of ET


Embryogenic cell line (ECL) 2 58,053.39 15.71 \0.001 50,344.79 17.17 \0.001

Maturation medium (M) 3 60,029.64 19.25 \0.001 46,334.89 15.80 \0.001

ECL 9 M 6 22,196.51 6.01 \0.001 19,909.98 6.79 \0.001

Error 24 3,694.79 2,932.41

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

0

200

400

600

800

1000

H 4 9

H 5 8

H 1 5 0

H 6 0

H 4 5

H 3 2

H 4 7

H 1 9 5

H 3 0

H 2 3 9

H 2 1 7

H 1 5 3

H 2 9

N º o f m a t u r e s o m a t i c e m b r y o s p e r g r a m o f

e m b r y o g e n i c t i s s u e ( F W )

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 embryo-

genic tissue (ET) in Pinus halepensis (M ± SE)

ECL Germinated somatic embryosper gram (FW) of ET

H29 270.0 ± 52.8a

H30 30.0 ± 10.9c

H32 32.3 ± 11.7c

H45 19.5 ± 9.0c

H47 30.2 ± 15.5c

H49 4.0 ± 4.0c

H58 10.0 ± 8.0d

H60 11.0 ± 6.2c

H150 0.0 ± 0.0

c

H153 101.8 ± 20.6bc

H195 58.2 ± 15.8c

H217 171.0 ± 32.9ab

H239 42.6 ± 11.0c

The somatic embryos were germinated on half strength LP (Quoirinand Lepoivre 1977, modified by Aitken-Christie et al. 1988) sup-plemented with 2 g L-1 of activated charcoal

Means followed by the same letter (a, b, c or d) are not significantlydifferent at p \ 0.05 Tukey’s post hoc test

Trees (2013) 27:1339–1351 1347

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(Krajnaková 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 initi-

ation and proliferation suggest that a multi-step protocol

would be better. Montalbán e t a l . (2012b) described a

protocol to induce organogenesis in P. radiata buds fromadult trees and containing two steps, one in presence of

high cytokinin concentration and another in absence of

plant growth regulators. Furthermore, Fernández-Guijarro

et al. (1995) in Quercus suber SE reduced the high con-

centrations 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

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

(Montalbán et al. 2010) o r in P. pinaster (Lelu-Walteret 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 (Lelu-

Walter et al. 2008) where the effect of AC depended on

the line tested. AC plays an essential role in micro-

propagation 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

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

ECL Germinated somatic embryos per gram of ETMaturation media

Mean

Low sucrose High sucrose

Low glutamine High glutamine Low glutamine High glutamine

A B C D

h19 28.6 ± 14.3c

66.7 ± 26.5bc

33.3 ± 33.3bc

52.4 ± 17.2bc

45.2 ± 11.2b

h41 72.7 ± 8.6bc

64.1 ± 19.6bc

188.0 ± 40.8b

21.4 ± 11.3c

86.5 ± 21.2b

h42 97.8 ± 19.4bc 115.6 ± 49.5bc 400.0 ± 61.1a 75.6 ± 24.8bc 172.2 ± 43.8a

Mean 66.3 ± 12.5b

82.1 ± 19.1b

207.1 ± 58.0a

49.8 ± 12.2b

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 Tukey’s post hoc test. In the last row and the last column appear the mean for the number of somatic embryos (%) onthe 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 Tukey’s post hoc test

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 High glutamine Low glutamine High glutamine

A B C D

h19 28.6 ± 14.3c 81.0 ± 26.5bc 47.6 ± 47.6c 71.4 ± 16.5bc 57.1 ± 14.0b

h41 89.7 ± 7.4bc 68.4 ± 23.8c 247.9 ± 52.5b 51.3 ± 31.2c 114.3 ± 27.9b

h42 115.6 ± 27bc 142.2 ± 49.5bc 435.6 ± 54.1a 88.9 ± 17.8bc 195.6 ± 45.5a

Mean 78.0 ± 15.8b

97.2 ± 21.0b

243.7 ± 61.6a

70.5 ± 14.4b

The ET was cultured on different maturation media (A, B, C and D). Different letters show significant differences at p \ 0.001 by Tukey’s 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 Tukey’s post hoc test

1348 Trees (2013) 27:1339–1351

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some of them and detrimental in others (Montalbán 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 maturationmedium) 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 morpho-

logically 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 alower 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) (Mont-

albán et al. 2010). However, as Montalbán 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 prolif-

eration and maturation of somatic embryos in different

species (Pérez-Rodrı́guez 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 (Pérez-Rodrı́guez et al. 2006).

In our experiment, in the most productive lines, the mat-

uration 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

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 Aitken-

Christie 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 Percyet al. (2000) in P. monticola. Although germination has

been accomplished in other Pinus species with a post-

maturation treatment (Maruyama and Hosoi 2012) or on

media without AC (Klimaszewska et al. 2001), this ger-

mination procedure and medium has been proven to be

suitable for micropropagation in species such as P. radiata

(Montalbán et al. 2010; Montalbán 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 Tec-

nologı́a—Spain (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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