ORIGINAL PAPER

Plant regeneration in Stone pine (Pinus pinea L.) by somaticembryogenesis

E. Carneros Æ C. Celestino Æ K. Klimaszewska ÆY.-S. Park Æ M. Toribio Æ J. M. Bonga

Received: 21 May 2008 / Accepted: 13 May 2009

� Springer Science+Business Media B.V. 2009

Abstract Regeneration of plants by somatic embryo-

genesis (SE) was achieved in Stone pine (Pinus pinea), one

of the most characteristic tree species of the Mediterranean

ecosystem. The initial explants were megagametophytes

containing zygotic embryos from five selected half-sib

families collected at different dates over 2 consecutive

years. Rates of extrusion and initiation of SE differed in

both years. However, qualitative patterns were very simi-

lar: for most families, the responsive developmental win-

dow was from late cleavage polyembryony to early

cotyledonary stage. The highest overall mean frequencies

of extrusion and SE initiation (7 and 0.9%, respectively, for

the five families and the eight 2006 collections) were

obtained on a modified Litvay’s medium with 9 lM 2,4-D

and 4.5 lM BAP, supplemented with L-glutamine and

casein hydrolysate. Families showed large differences in

frequencies of SE initiation from year to year. Only seven

embryogenic lines were induced in 2005, representing

three of the five families tested, whereas 34 lines from all

the families were obtained in 2006. Proliferation of

embryonal masses (EM) was significantly improved when

they were subcultured after dispersing in liquid medium

and collected on filter paper disks, instead of being sub-

cultured as small clumps. This effect showed a significant

interaction with genotype. Several preconditioning treat-

ments and culture media combinations were tested for

embryo development and maturation. The high prolifera-

tion rate of EM hampered somatic embryo development.

However, up to 42 mature embryos from different lines of

three of the five families were obtained, 23 of them ger-

minated and seven converted into somatic seedlings.

Keywords Conifer � Embryonal mass � Genetic effect �Half-sib families � Somatic embryogenesis �Somatic seedling � Tree breeding

Abbreviations

2,4-D 2,4-Dichlorophenoxyacetic acid

ABA Abscisic acid

AC Activated charcoal

BAP Benzylaminopurine

EM Embryonal mass

FW Fresh weight

MVF Multi-varietal forestry

OP Open-pollinated

PGR Plant growth regulators

PPFD Photosynthetic photon flux density

SE Somatic embryogenesis

se Standard error

Introduction

Domestication of forest tree species is very recent com-

pared with agricultural crops. For centuries, forestry has

E. Carneros � C. Celestino � M. Toribio (&)

Instituto Madrileno de Investigacion y Desarrollo Rural, Agrario

y Alimentario (IMIDRA), Apartado 127, Alcala de Henares,

28800 Madrid, Spain

e-mail: mariano.toribio@madrid.org

K. Klimaszewska

Natural Resources Canada, Canadian Forest Service, Laurentian

Forestry Centre, P.O. Box 10380, Stn. Sainte-Foy, Quebec,

QC G1V 4C7, Canada

Y.-S. Park � J. M. Bonga

Natural Resources Canada, Canadian Forest Service, Canadian

Wood Fibre Centre, P.O. Box 4000, Fredericton, NB E3B 5P7,

Canada

123

Plant Cell Tiss Organ Cult

DOI 10.1007/s11240-009-9549-3

been more a ‘‘mining’’ than a ‘‘farming’’ activity because,

among various reasons, of the biological attributes of forest

trees—mainly their long generation times and, for most

species, poor ability to propagate vegetatively. This makes

forest genetic improvement programs much more difficult

than their agricultural counterparts. In particular, cloning,

which has been used for centuries in several woody crops

and fruit trees to capture all the components of genetic

variance, is routinely applied to only a few species of the

genus Cryptomeria, Populus, Salix, and Eucalyptus.

However, modern biotechnology is providing new tools in

addition to conventional plant breeding for forest tree

improvement (El-Kassaby 2004). Of these various tools,

somatic embryogenesis (SE) is considered the best vege-

tative propagation tissue culture method for most tree

species, both hardwood and softwood (Merkle and Nairn

2005; Nehra et al. 2005). This vegetative propagation

technique is enabling the development of multi-varietal

forestry (MVF), the use of tested tree varieties in forestry

(Park et al. 2006).

Plant regeneration by SE has been obtained in several

Pinus species (Zoglauer et al. 2003), but to our knowledge

it has not been reported for Stone pine (Pinus pinea L.).

Stone pine is one of the most important tree species in the

Mediterranean ecosystem, and is used for different pur-

poses, such as environmental restoration (afforestation of

coastal areas and continental dunes) and ornamental

planting. However, the most distinctive feature of this

species is the large size of its megagametophytes, which

are full of nutrients and therefore highly prized gastro-

nomically. In Spain, Stone pine covers around 360,000 ha,

which represents about 75% of the species’ total area.

Spain’s breeding program is mainly focused on improving

seed (pine nut) yield (Mutke et al. 2000). As the most

important economical use of this forest species is as a

‘‘fruit tree,’’ the development of clonal rootstocks to which

scions from trees with a high annual nut production can be

grafted (grafted varieties) is of great importance.

Vegetative propagation by rooting of cuttings in Stone

pine is difficult, and problems related to cyclophysis and

topophysis effects usually appear. Regarding micropropa-

gation, there are several reports describing the induction of

adventitious shoots from embryonic explants, and even

rooting of these microcuttings (Capuana and Giannini

1995; Diamantoglou et al. 1990; Garcıa-Ferriz et al. 1994;

Gonzalez et al. 1998; Oliveira et al. 2003; Valdes et al.

2001). However, the advantages of SE—particularly

maintenance of regeneration potential by cryopreservation

while the testing of clones is in progress (Park 2002)—

justify the development of SE protocols for this species.

Somatic embryogenesis is defined as a multi-step

regeneration process starting with formation of embryo-

suspensor masses, followed by embryo formation,

maturation, and plant regeneration. The induction of the

embryogenic response is influenced by several factors,

including basal medium components, exposure to exoge-

nous plant growth regulators (PGR), and culture condi-

tions. However, the features of the initial explant, mainly

the type and developmental stage as well as the genetic

make-up, largely determine the success of the embryogenic

response. Except in a few cases (Lelu et al. 1999), auxins

and cytokinins are necessary for inducing and sustaining

embryogenic tissue proliferation, whereas embryo early

development is triggered by the withdrawal of PGR. Fur-

ther growth of early somatic embryos to mature forms

usually requires the presence of abscisic acid (ABA) (von

Arnold et al. 2002).

For most coniferous species, especially those of the

genus Pinus, immature embryos are the optimal material

for induction of SE (see reviews by Gupta and Grob 1995;

Klimaszewska and Cyr 2002; Lelu-Walter et al. 2006;

Pullman and Johnson 2002; Tautorus et al. 1991; von

Arnold et al. 2002). Moreover, the embryogenic compe-

tence is often restricted to a short yearly time window, and,

therefore, identification of the proper developmental stage

of the immature zygotic embryo to initiate an embryogenic

line is of paramount importance. Nevertheless, somatic

embryos could also be induced sporadically from mature

zygotic embryos of a few pine species, increasing the

availability of suitable explants (Deb and Tandon 2002;

Garin et al. 1998; Radojevic et al. 1999; Tang et al. 2001).

Genotype plays a substantial role in morphogenic

responses. Many papers have reported large variation in SE

initiation frequencies among families of different conifer

species (Cheliak and Klimaszewska 1991; Garin et al.

1998; Miguel et al. 2004; Pullman and Johnson 2002).

Furthermore, detailed studies revealed a variable degree of

genetic control of the different stages of plant regeneration

by SE. Among them, initiation has been shown to be under

strong genetic control, mainly additive in both Picea gla-

uca and Pinus taeda (MacKay et al. 2006; Park et al.

1993).

The main objective of this work was to define methods

to achieve SE in Stone pine, by determining the develop-

mental window in seeds during which induction is feasible,

and considering the influence of culture medium and

genetic effects for the refinement of the SE process.

Materials and methods

Plant material

Open-pollinated (OP) green cones were collected from

several ramets of five selected clones in a Stone pine clonal

bank established at the National Forest Breeding Centre

Plant Cell Tiss Organ Cult

123

‘‘Puerta de Hierro’’ in Madrid (Spanish Ministry of Envi-

ronment). In order to induce somatic embryogenesis they

were collected at about weekly intervals from 23 June to 27

July 2005, and from 7 June to 27 July 2006. Whole cones

were submerged in 70% (v/v) ethanol for 3 min, followed

by immersion in a 10% (v/v) solution of commercial bleach

(sodium hypochlorite: 3.5% active chlorine) for 10 min,

and then air dried and stored in paper bags at 4�C for a

maximum of 2 weeks while all seeds were dissected. For

culture initiation, seeds from each of the five half-sib

families (11, 47, 58, 70, and 88) were removed from the

cones and disinfested in 10% (v/v) commercial bleach with

one drop of Tween� 20 for 15 min, and rinsed three to four

times with sterile water. Then, seed coats were removed.

In order to determine the developmental stage of zygotic

embryos in each collection date, a few zygotic embryos

were excised from randomly selected megagametophytes

collected weekly from the last week of May of both years.

For the first three harvests starting on 7 June of both years

when embryos were at a very early stage, slide samples

were prepared after staining with 1% (w/v) acetocarmine

and microscopically examined. Later collections of both

years were macroscopically examined.

Initiation of somatic embryogenesis

For initiation of embryogenic cultures, three culture media

were tested in 2005 and five culture media, two of which

were common to both years, were tested in 2006.

In the experiment carried out in 2005, cones from the

five families were sampled at six collection dates (23 June,

1, 6, 13, 20, and 27 July). The basal medium, labeled as

mLV2, consisted of a modified Litvay’s (Litvay et al. 1985)

nutrient medium with half-strength macroelements, and

full-strength Fe-EDTA and vitamins. Microelements were

modified as follow: boric acid and manganese and zinc

salts were at full strength; molybdenum, copper, and iodine

salts were at half strength; cobalt salt was reduced to

0.01 mg l-1, and 0.72 mg l-1 NiCl2 was added. Three

combinations of growth regulators at different concentra-

tions were tested: low, L-mLV2 (2.4 lM 2,4-D and 2.2 lM

BAP); medium, M-mLV2 (9 lM 2,4-D and 4.5 lM

BAP); and high, H-mLV2 (15 lM 2,4-D and 4.5 lM

BAP) (Table 1). All media contained sucrose at 10 g l-1.

Overall, 3,500 explants were cultured (six collection

dates 9 five half-sib families 9 three culture media 9 ten

seeds per replicate 9 three to four replicates per

treatment).

In the experiment carried out in 2006, cones from the

same five families were collected on eight collection dates

(7, 14, 23 June and 1, 6, 13, 20, and 27 July). Five culture

media were used; two of them were the same as described

above: M-mLV2 (9 lM 2,4-D and 4.5 lM BAP) and H-

mLV2 (15 lM 2,4-D and 4.5 lM BAP). The third culture

medium, labeled as ultra-high UH-mLV2, was the same

basal medium but supplemented with 20 lM 2,4-D and

4.5 lM BAP. Two additional media, in which the basal

medium was a modified Litvay’s medium with half-

strength macroelements and full-strength microelements,

Fe-EDTA and vitamins (mLV) (Klimaszewska et al. 2001),

were also assayed. They contained PGR at medium, M-

mLV (9.5 lM 2,4-D and 4.5 lM BAP) and high, H-mLV

(15 lM 2,4-D and 4.5 lM BAP) levels (Table 1). These

later two media contained sucrose at 20 g l-1. Overall,

7,200 explants were cultured (eight collection dates 9 five

half-sib families 9 five culture media 9 ten seeds per

replicate 9 three to four replicates per treatment).

Before autoclaving, 1 g l-1 casein hydrolysate (Duchefa

Biochimie B V., The Netherlands) was added to all media,

the pH was adjusted to 5.8, and they were solidified with

4 g l-1 Gelrite� (G1910, Sigma-Aldrich Co., USA). Also,

all media were supplemented with a filter-sterilized solu-

tion of 0.5 g l-1 L-glutamine (G8540, Sigma-Aldrich Co.,

USA) added to the cooled medium.

Megagametophytes with enclosed zygotic embryos were

placed on 90 9 16 mm Petri dishes, each contain-

ing approximately 25 ml of medium. Ten explants per

dish, sealed with Parafilm� (American Can Company,

Table 1 Micronutrient modifications, and plant growth regulators and sucrose content in the culture media used for initiation and maintenance

of embryogenic tissues

Label Salts (mg/l) PGR (lM) Sucrose (g/l)

NiCl2 CoCl2 CuSO4 Na2MoO4 KI 2,4D BAP

UH-mLV2 0.72 0.01 0.25 0.63 2.1 20 4.5 10

H-mLV2 0.72 0.01 0.25 0.63 2.1 15 4.5 10

M-mLV2 0.72 0.01 0.25 0.63 2.1 9 4.5 10

L-mLV2 0.72 0.01 0.25 0.63 2.1 2.4 2.2 10

H-mLV – 0.125 0.5 1.25 4.15 15 4.5 20

M-mLV – 0.125 0.5 1.25 4.15 9.5 4.5 20

Plant Cell Tiss Organ Cult

123

Greenwich, CT), were cultured and kept in darkness at

23 ± 1�C for the duration of each experiment. The cultures

were examined weekly for tissue growth. The megaga-

metophytes were not subcultured during the experiment,

which lasted up to 12 weeks. Three or four replicates (Petri

dishes) per each treatment were arranged, depending on the

availability of megagametophytes.

Extrusion was recorded when zygotic embryos pushed

out of the micropylar end of the megagametophyte, and

proliferated to form an initial embryonal mass (EM).

Explants were considered to have initiated SE when the

tissue showed continuous growth and produced amounts

sufficient for subculture. The number of established lines

was recorded after 4 months of culture under conditions

described below.

Proliferation and maintenance of EM

After 4–12 weeks on initiation media, proliferating EM

were separated from the megagametophyte, and subcul-

tured biweekly onto fresh maintenance medium in darkness

at 23 ± 1�C. The maintenance medium was the M-mLV

medium described above and was used for all embryogenic

lines, regardless of the culture medium used for initiation.

The effect of two procedures on EM proliferation was

studied. In the ‘‘clump procedure,’’ small pieces of tissue

collected at the EM periphery from different embryogenic

lines were subcultured under the maintenance conditions

described above. Initial explants were individual clumps of

about 100 mg of fresh weight. At the beginning of the

treatment, one clump was cultured per 90 mm diameter

Petri dish. Five dishes (replicates) per embryogenic line

were arranged. After each biweekly subculture, the indi-

vidual clumps were divided into four pieces and placed

again onto five new Petri dishes per line.

In the ‘‘dispersal procedure’’ (Klimaszewska and Smith

1997; Lelu-Walter et al. 2006), about 100 mg samples,

collected at the EM periphery, were suspended in 10 ml of

liquid M-mLV medium, vigorously shaken to break up the

tissue into a fine suspension, and poured onto a filter paper

disk (80 g/m2, 60–68 lm pore, Filter-Lab, ANOIA; Bar-

celona, Spain) in a Buchner funnel. Low-pressure pulse

was applied to drain the liquid, and the filter paper with

attached cells was placed on the surface of fresh M-mLV

semi-solid medium in a 90 mm diameter Petri dish. Five

filter paper disks with embryogenic tissue were arranged in

five dishes (replicates) per line and subcultured biweekly.

Five embryogenic lines chosen from different families

(7F11 and 2F70 initiated in 2005; 2F47, 9F58 and 6F88

initiated in 2006) underwent both procedures to study the

effect of procedure and genotype. Growth rate of tissue in

each replicate was recorded after 2, 4, and 6 weeks in

darkness at 23 ± 1�C. To calculate growth rate, fresh

weight of EM was determined at the beginning of each

subculture by subtracting the weight of a fresh medium-

containing dish from the weight of the same fresh medium-

containing dish but including the embryogenic tissue. The

dependent variable was the relative fresh weight increa-

se(s) after each period, defined as the fresh weight at the

end of that period minus the initial fresh weight, divided by

the initial fresh weight.

Maturation of somatic embryos

Several randomly chosen embryogenic cell lines from

different families were used to determine their capacity to

produce mature somatic embryos. The ages of the cultures

ranged from 1 to 10 months from the onset of initiation.

Two preconditioning procedures with several maturation

treatments were tested based on previous reports (Lelu

et al. 1999; Klimaszewska et al. 2001; Bonga 2004; Lelu-

Walter et al. 2006).

In the first experiment, carried out in 2006, four estab-

lished embryogenic lines (7F11, 13F58 and 2F70 initiated

in 2005; 5F47 initiated in 2006) were preconditioned by

subculturing EM on UL-mLV medium that was made up of

mLV medium with reduced PGR concentrations (0.24 lM

2,4-D and 0.22 lM BAP) with 20 g l-1 sucrose and

4 g l-1 Gelrite, for 1 month with biweekly subculture.

Then, a suspension of embryogenic tissue at a concentra-

tion of 10 g FW of EM per liter was prepared with the

same medium used for preconditioning but lacking gelling

agent, PGR, and glutamine, and supplemented with

10 g l-1 of activated charcoal (AC, C6289, Sigma-Aldrich

Co., USA). The dispersed tissue was collected by pouring

5 ml of the suspension onto filter paper disks as previously

described, and cultured on the different maturation media.

The maturation treatments were carried out according to a

complete factorial design in which the effects of nutrient

medium, AFC medium (Bonga 2004) vs. mLV; type of

sugar, maltose vs. sucrose both at 60 g l-1; gelling agent,

20 g l-1 Plantagar� (S1000, B&VS.R.L., Italy) vs. 10 g l-1

Gelrite; and ABA concentrations (80, 121, and 161 lM)

were tested. Therefore, considering the four genotypes, 96

treatments were included in the experiment. Cultures were

not transferred to fresh medium for the duration of the

experiment.

In the second experiment, carried out in 2006, seven

embryogenic lines (7F11, 13F58 and 2F70 initiated in

2005; 2F47, 5F47, 9F58 and 2F88 initiated in 2006) were

tested. The preconditioning of the EM was carried out as

described above, but all components of the culture med-

ium, except for PGR and Gelrite, were reduced by half.

Then, the suspension in liquid medium lacking PGR and

glutamine was prepared as above, but with the half-strength

mLV medium, and with or without 10 g l-1 of AC. Five

Plant Cell Tiss Organ Cult

123

milliliters of the suspensions (50 mg FW) were poured

onto filter paper disks and placed on the half-strength mLV

medium without PGR, solidified with 4 g l-1 Gelrite and

with or without 10 g l-1 of AC, for 1 week. Disks with

tissues from suspension prepared with AC were cultured on

medium without this compound, and vice versa. Matura-

tion was performed on mLV medium supplemented with

60 g l-1 sucrose and 10 g l-1 Gelrite. Two ways of sup-

plying ABA, continuous vs. pulse, at two concentrations,

80 and 121 lM, and 121 and 200 lM, respectively, were

tested. In total, 56 treatments were performed. Continuous

supply was accomplished by maintaining the paper disks

with EM on the ABA-containing media for the whole

length of the experiment, with monthly subcultures. In the

pulse treatments, cultures were on the ABA containing

media for 7 days, and then were transferred to PGR-free

medium for the whole length of the experiment, also with

monthly subcultures.

In both maturation procedures, cultures were kept in

darkness at 23 ± 1�C, and the treatments lasted 16 weeks.

Each filter paper disk with 50 mg FW of EM placed into a

90-mm diameter Petri dish with maturation medium was

considered a replicate, and five replicates per treatment

were arranged. The pH of the media was adjusted to

5.8 before autoclaving. Solutions of ABA and glutamine

were filter sterilized and added to the cooling autoclaved

medium.

Maturation ability of the different treatments was

recorded as the number of morphologically normal somatic

embryos (opaque, presence of cotyledons, embryonal root

caps, and smooth hypocotyls) obtained from 250 mg FW

of EM, per embryogenic line within each treatment.

Embryo germination and conversion to plant

Mature somatic embryos were picked from filter paper

disks after 4–6 months on maturation media and placed

horizontally, all with the cotyledons facing in one direc-

tion, on medium for germination. This medium consisted

of AFC nutrient medium without PGR, supplemented with

30 g l-1 sucrose and 6 g l-1 Plantagar. Petri dishes

(60 9 16 mm) were filled with 10 ml of this semisolid

medium. For pre-germination treatment, embryos were

stored in darkness at 4�C for 1 month. Then, cultures were

transferred to a growth chamber at 23 ± 1�C, with a 16-h

photoperiod and photosynthetic photon flux density

(PPFD) of 17–20 lmol m-2 s-1 provided by mixed Syl-

vania Gro-lux and Philips cool-white fluorescent tubes for

1 month. Petri dishes were tilted to a slanted position at an

angle of approximately 408, and embryos positioned with

their embryonal root caps pointing downward. When

somatic embryos started to germinate, they were asepti-

cally transplanted for further growth to culture tubes ‘‘De

Wit’’ (W1607, Duchefa Biochemie 2003) filled with 15 ml

liquid AFC medium without organic ammonium and PGR,

with 10 g l-1 sucrose and sterile perlite (2 mm particle

size) as support. The developing somatic seedlings were

placed in a growth chamber at 23 ± 1�C, and exposed to

PPFD of 120–180 lmol m-2 s-1 for a 16-h photoperiod.

After the plumule reached 1 cm, plantlets were trans-

planted ex vitro to 240 ml forest containers filled with

substrate (peat: perlite, 3:1, v/v) and the slow release fer-

tilizer Osmocote� at 3 g l-1 for acclimatization. They

were placed in a growth chamber at 23 ± 1�C, under 80%

relative humidity, and a 16-h photoperiod with PPDF of

120–180 lmol m-2 s-1.

Statistical analysis

Effects of crop year, collection date, family, culture med-

ium, genotype, and maintenance culture procedure were

evaluated by one- or two-way ANOVA, using Statistica for

Windows 5.1 software (StatSoft, Tulsa, OK). Data on fre-

quency of extrusion and initiation of embryogenic lines

were transformed by arcsin H(%). Relative growth data of

EM were transformed using the normal logarithm. Mean

values are shown with standard errors (se). Significant

differences between means were determined by Duncan’s

test at a significance level of 5%.

Results

Developmental stage of the zygotic embryos

Early observations showed that megagametophytes col-

lected in May of both years were translucent with visible

archegonia, but cell division of a developing proembryo

was not detected. We observed little variation in the

developmental stage of zygotic embryos for the same

collection dates between the 2 years. However, immature

embryos collected on 23 June 2005 were slightly more

advanced in development than on the same date in 2006,

although dominance was not detected yet.

On 7 June of both years, megagametophytes were

slightly opaque (Fig. 1a) and early cell division of the

proembryo was noticed (pre-cleavage stage) (Fig. 1b). On

14 June, megagametophytes were opaque (Fig. 1c) and

most embryos were at the beginning of the cleavage stage

(Fig. 1d), with the terminal embryonal cells and subter-

minal suspensor cells placed deeper inside the female

gametophyte (Fig. 1c). On 23 June, cleavage polyembry-

ony was evident (Fig. 1e, f). At the first collection date in

July, embryos were at the post-cleavage stage, and the

beginning of dominance could be observed (Fig. 1g). On 6

July, the development of the dominant embryo occurred,

Plant Cell Tiss Organ Cult

123

showing the establishment of polarity, and the subordinate

embryos aborted (Fig. 1h). By mid-July, embryo domi-

nance was well established and the zygotic embryos

entered the precotyledonary stage, maintaining long sus-

pensors (Fig. 1i). At the last collection dates, on 20 and 27

July, completely developed embryos with elongating

cotyledons that were attached to remaining suspensor cells,

could be observed (Fig. 1j, k).

Extrusion and initiation of SE

In the experiments carried out in both years, embryogenic

tissue began to grow from the micropylar end of the

megagametophytes between 4 and 12 weeks after plating

(Fig. 2a). Some explants exhibited proliferation of muci-

laginous white tissue, which was often accompanied by

rupture of the megagametophyte along its longitudinal axis

(Fig. 2b). Previous experiments had shown that, although

many megagametophytes showed protrusion of this putative

embryogenic tissue, only a part of those proliferations could

be maintained in successive subcultures. Therefore, extru-

sion and SE initiation responses were recorded separately.

For all families, culture media and collection dates in

2005, 152 cultures of extruded tissues were obtained, rep-

resenting 4.3% of the 3,500 explants cultured. However, only

seven embryogenic lines were established, representing only

Fig. 1 Developmental stages of

Pinus pinea zygotic embryos

according to collection date

a June 7th, 2006.

Megagametophyte enclosing a

zygotic embryo with primary

suspensor (bar = 1 mm).

b June 7th, 2006. Zygotic

embryo with four tiers with four

cells each (arrow) stained with

acetocarmine (bar = 0.2 mm).

c June 14th, 2006. Embryo with

suspensor inside corrosion

cavity (bar = 1 mm). d June

14th, 2006. Embryos at the

beginning of cleavage stained

with acetocarmine

(bar = 0.1 mm). e June 23rd,

2006. Embryos at the cleavage

polyembryony stage

(bar = 0.5 mm). f June 23rd,

2006. Embryos at the cleavage

polyembryony stage stained

with acetocarmine

(bar = 0.1 mm). g July 1st,

2006. Development of dominant

embryo (bar = 0.25 mm).

h July 6th, 2006. Dominant

embryo with established

polarity (bar = 0.5 mm). i July

13th, 2006. Precotyledonary

embryo: development of shoot

apical meristem

(bar = 0.5 mm). j, k July 20–

27th, 2006. Elongating

cotyledonary embryos inside

megagametopytes

(bar = 1 mm)

Plant Cell Tiss Organ Cult

123

0.2% of the initial explants. In 2006, 406 cultures of extruded

tissues were produced, representing 5.6% of the 7,200 cul-

tured explants, and 34 embryogenic lines were established,

representing 0.5% of the initial explants. Therefore, explants

were more responsive in 2006 than in 2005. When data from

media that were common to both years were considered, a

significant effect of year of collection was observed on both

extrusion (4.8 and 8.1% in 2005 and 2006, respectively,

P = 0.000) and SE initiation (0.3 and 0.6% in 2005 and

2006, respectively, P = 0.042).

Regarding the influence of culture medium, a significant

effect on the frequency of initiation was detected in the

2006 experiment (P = 0.046). This was mostly due to

basal medium composition, because mLV2 media rendered

frequencies that were three to four times higher than those

of mLV media (Table 2). However, 2,4-D concentrations

in the range of 9–20 lM did not affect either extrusion or

initiation in both years. Best results were observed with

M-mLV2 medium in 2006 that gave a frequency of initi-

ation nine times higher than obtained in 2005. However,

H-mLV2 medium with increased 2,4-D concentrations per-

formed equally in 2005 and 2006 experiments (Table 2).

The family 9 culture medium interaction was not

significant.

Fig. 2 Somatic embryogenesis in Pinus pinea. a Extrusion of putative

embryogenic tissues from a megagametophyte containing a developing

dominant zygotic embryo collected July 1st, 2006 (bar = 2 mm).

b Extrusion of putative embryogenic tissues from a cotyledonary zygotic

embryo collected July 20th, 2006 (bar = 2 mm). c Embryogenic tissue

growing on maintenance medium as clumps (bar = 4 mm). d Embryo-

genic tissue growing on maintenance medium dispersed on paper disks

(bar = 5 mm). e Early developmental stage of somatic embryos

growing in clumps (bar = 1 mm) f Early development of somatic

embryos growing dispersed on disks (bar = 0.1 mm). g Embryogenic

culture showing developing somatic embryos (bar = 0.5 mm).

h Maturation of somatic embryos in AFC medium with 121 lM ABA,

using activated charcoal in close contact with embryogenic tissue

(bar = 5 mm). i–k Different developmental stages of cotyledonary

somatic embryos (bar = 0.5 mm). l Germination of somatic embryo

(bar = 2 mm). m Somatic seedlings growing in perlite moistened with

liquid medium (Culture tube maximum diameter = 27 mm). n Accli-

matized somatic seedlings in forest containers (Container maximum

diameter = 52 mm)

Plant Cell Tiss Organ Cult

123

Although mLV2 medium was best suited for initiation,

preliminary trials showed that some embryogenic lines

gradually declined when they were subcultured in the same

initiation medium, producing slightly darker and less vig-

orously growing tissue. This decline was not observed when

the M-mLV medium was used. Therefore, all embryogenic

lines obtained from the several initiation media, were sub-

sequently subcultured on mLV medium supplemented with

the lower PGR concentrations for maintenance (9.5 lM

2.4D and 4.5 lM BAP). When the established embryogenic

lines were subcultured on growth regulator-free medium,

EM did not proliferate (data not shown).

The ability of explants to initiate extrusion was signifi-

cantly influenced by the collection date of the cones in the

experiments carried out both in 2005 (P = 0.009) and 2006

(P = 0.000). However, a statistically significant effect on

SE initiation was not detected. The extrusion frequency had

almost identical patterns in both years. It was zero or very

low on the first collection dates, increasing until the end of

June, peaking during the first week of July when zygotic

embryos were at the post-cleavage stage, and declining

until mid July, when most of the zygotic embryos started to

develop cotyledons (Fig. 3). Then, although not statisti-

cally significant, higher values of extrusion frequency were

recorded at the subsequent collection dates. The decline in

mid July was mainly due to the absolute lack of response of

families 58 and 70 in the 2005 experiment, and family 58

in 2006. The frequency of SE initiation showed similar

patterns in both years. The maximum values were reached

at the beginning of July, but with a slight delay in the year

2005 (Fig. 3).

The OP (half-sib) family effect on the frequency of

established lines was close to the 5% significance level, in

both 2005 (P = 0.053) and 2006 (P = 0.062), when data

from all collection dates and culture media were analyzed.

Large variation in the mean frequency of SE initiation

among the OP families was observed (Fig. 4). In the 2005

experiment, explants from families 47 and 88 did not

respond at all, only one embryogenic line could be estab-

lished from each of families 58 and 70, and five lines were

generated from family 11. In the 2006 experiment, six lines

were obtained from family 11, six from family 47, twelve

from family 58, one from family 70, and nine from family

88. Although the interaction family 9 collection date was

not significant, when looking at the behaviour of each

individual family along all the collection dates, very dif-

ferent patterns of frequency of initiation could be noticed

(Fig. 5). Although most families generally showed a rela-

tively narrow ‘‘window of response,’’ family 11, which was

the only family that responded equally in 2005 and 2006

(0.7 and 0.4% initiation not significantly different, and 5

and 6 established embryogenic lines, respectively), had a

broader range of responsive period (Fig. 5).

Out of 10,700 megagametophytes tested, 558 prolifer-

ating cultures were initiated on different culture media in

the 2005–2006 experiments, but only 7.3% of them formed

Table 2 Initiation of somatic embryogenesis in Pinus pinea

Medium 2005 2006

UH-mLV2 non-tested 0.7 ± 0.2b

H-mLV2 0.4 ± 0.2a 0.4 ± 0.2ab

M-mLV2 0.1 ± 0.1a 0.9 ± 0.3b

L-mLV2 0.1 ± 0.1a non-tested

H-mLV non-tested 0.1 ± 0.1a

M-mLV non-tested 0.3 ± 0.1ab

Effect of culture medium on the frequency (%) of initial zygotic

embryos forming established embryogenic lines. Values are mean-

s ± se from 117 and 144 replicates, of 7–10 seed per replication,

depending on the year (five families, and six and eight collection data

in crop years 2005 and 2006, respectively)

Means within the same column followed by the same letter are not

significantly different at P = 0.05 (Duncan’s multiple range test)

0

2

4

6

8

10

12

14

Collection date

% E

xtru

sion

2005

2006

0

0,5

1

1,5

2

2,5

7.6 14.6 23.6 1.7 6.7 13.7 20.7 27.7

7.6 14.6 23.6 1.7 6.7 13.7 20.7 27.7

Collection date

% I

niti

atio

n

2005

2006

A

B

Fig. 3 Effect of collection date on the frequency of a extrusion and binitiation of embryogenic lines from developing zygotic embryos of

Pinus pinea, in the 2005–2006 crop years. Each point represents the

mean response of the five OP families in the two common culture

media tested in both years. Values ± se are means of 34–40

replications in 2005 and 27–42 replications in 2006, of 7–10 seeds

per replication

Plant Cell Tiss Organ Cult

123

established embryogenic lines (seven in 2005 and 34 in

2006) over a period of more than 6 months. These lines

were obtained from all the tested families. Samples of the

established embryogenic lines were cryopreserved (data

not shown).

Proliferation and maintenance of EM

As previously stated, the EM were subcultured on M-mLV

medium for maintenance. Tissue dispersal increased the

growth of lines. When the EM were separated into small

pieces and subcultured (Fig. 2c), their growth rate was

enhanced and 16 clumps per initial clump were generated

after two biweekly subcultures. However, when they were

dispersed on filter paper disks (Fig. 2d), proliferation was

even more improved (Fig. 6). The growing EM was com-

posed of proembryogenic and early embryogenic structures

with long suspensors, which gave it a characteristic spiky

appearance (Fig. 2e, f).

After three biweekly subcultures, there was no signifi-

cant difference between the growth of EM initiated in 2005

and initiated in 2006, both for the clump procedure and for

the dispersal procedure. However, significant effects of

genotype (P = 0.002), method of proliferation (i.e., clump

vs. dispersal) (P = 0.000) and their interaction (P =

0.000) for relative FW increase of EM were found. Overall,

culturing 100 mg of fresh EM as small clumps resulted in

about 30-fold increase in FW after three biweekly sub-

cultures, whereas culturing the same using the dispersal

procedure increased up to about 70-fold (Fig. 6). The

interaction of the dispersal procedure with genotype was

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

1,1

11 47 58 70 88

Family

% In

itia

tio

n2005

2006

0 0 5 1 16 6 9112

Fig. 4 Effect of OP family on the frequency of initiation of

embryogenic lines from developing zygotic embryos of Pinus pinea.

Values represent means of data from all tested culture media and

collection data in the crop years 2005 and 2006. Values ± se are

means of 68–72 replications in 2005 and 134–159 replications in

2006, of 7–10 seeds per replication. The number of established

embryogenic lines obtained per family and collection year is shown at

the base of each column

Family 11

-0,5

0

0,5

1

1,5

2

2,5

3

3,5

Collection date

% I

niti

atio

n

Family 47

-0,50

0,51

1,52

2,53

3,54

4,5

Collection date

% I

niti

atio

n

Family 58

-0,5

0,5

1,5

2,5

3,5

4,5

Collection date

% I

niti

atio

n

Family 70

-0,5

0

0,5

1

1,5

Collection date

% I

niti

atio

n

Family 88

-0,5

0,5

1,5

2,5

3,5

4,5

7.6 14.6 23.6 1.7 6.7 13.7 20.7 27.7 7.6 14.6 23.6 1.7 6.7 13.7 20.7 27.7

7.6 14.6 23.6 1.7 6.7 13.7 20.7 27.7 7.6 14.6 23.6 1.7 6.7 13.7 20.7 27.7

7.6 14.6 23.6 1.7 6.7 13.7 20.7 27.7

Collection date

% I

niti

atio

n

A B

C D

E

Fig. 5 Effect of collection date

on the frequency of initiation of

embryogenic lines from

developing zygotic embryos of

Pinus pinea in crop year 2006,

for each one of the tested OP

families (a–e). Data are means

from all culture media. Each

point represents a mean ± se of

9–16 replications of 7–10 seeds

per replication

Plant Cell Tiss Organ Cult

123

also noteworthy. In the most extreme case, genotype 6F88,

when EM were subcultured as clumps, the initial FW after

the three biweekly subcultures was multiplied by about

eight, whereas using dispersal on filter disks, it was mul-

tiplied by about 96 (Fig. 6).

Maturation of somatic embryos

A few mature embryos were obtained from the two sets of

experiments. Differentiation of somatic embryos happened

on ABA-containing media (Fig. 2h) and followed a process

of maturation that resembled that of their zygotic coun-

terparts (Fig. 2g, and i, k).

When the first procedure of preconditioning was used,

only nine mature somatic embryos were obtained from all

the embryogenic lines and the 24 combinations of matu-

ration media. The best was line 5F47, which produced

seven mature embryos on the combination AFC basal

medium with maltose, agar, and 121 lM ABA. One

embryo was also obtained in the same line and medium,

but supplied with 161 lM ABA. One additional embryo

was obtained in line 13F58 cultured on AFC basal medium

with sucrose, agar, and 80 lM ABA. Therefore, mature

embryos were not obtained on any of the different mLV

based combinations, nor on AFC with Gelrite.

When the second procedure of preconditioning was

used, mature somatic embryos arose on mLV medium

(Table 3). On the other hand, when embryogenic tissue was

mixed with AC, four to five times as many mature embryos

were produced than when AC was in the culture medium.

In addition, continuous culture on ABA-containing med-

ium gave four to five times as many mature embryos than

ABA-pulse treatments (Table 3). The highest number of

responsive genotypes (four out of seven tested genotypes)

was obtained when tissue was mixed with AC and con-

tinuously cultured on 121 lM ABA-containing medium

(Table 3). No precocious germination of mature somatic

embryos was observed.

Germination and conversion into plants

All embryos obtained from both maturation experiments

were treated for germination. Embryos from the first

experiment of maturation did not germinate at all. How-

ever, 23 embryos out of the 33 mature embryos that were

obtained after using the second procedure of precondi-

tioning, germinated (Fig. 2l). Epicotyl growth occurred in

seven of these embryos, which converted into plants

(Fig. 2m, n).

0

10

20

30

40

50

60

70

80

90

100

110

7F11 2F47 9F58 2F70 6F88

Subcultures (s1,s2 & s3) and embryogenic line

Rel

ativ

e fr

esh

wei

ght

incr

ease

Clump

Disc

s3s2 s3s2s1 s1s2 s3s1s2 s3s1s2 s3s1

Fig. 6 Effect of subculture procedure and genotype on the accumu-

lated relative fresh weight increase of proliferating embryonal masses

established from zygotic embryos of Pinus pinea, along three

biweekly subcultures onto fresh semi-solid medium. Data represent

means ± se of 5 replicates of tissue clumps (black) and dispersed

tissue on paper disks (white) of five embryogenic lines from different

families, taken after 2 (s1), 4 (s2) and 6 (s3) weeks on maintenance

medium. Notation of embryogenic lines (7F11 and 2F70 initiated in

2005; 2F47, 9F58 and 6F88 initiated in 2006) includes the family that

they belong to

Table 3 Effect of activated charcoal (AC) during the preconditioning of embryogenic tissue for maturation, and way of application and

concentration of ABA during maturation, on the number of Pinus pinea mature somatic embryos per responsive embryogenic line (indicated

within parenthesis)

Preconditioning No. mature somatic embryos/responsive embryogenic line

ABA-continuous ABA-pulse Combined total

80 lM 121 lM Total 121 lM 200 lM Total

Tissue in close contact with AC 3 (5F47)

9 (9F58)

2 (5F47) 21 1 (5F47)

2 (9F58)

1 (5F47)

2 (9F58)

6 27

3 (2F47)

3 (9F58)

1 (2F70)

AC in the medium 1 (5F47) 1 (5F47) 6 0 0 0 6

2 (9F58) 2 (9F58)

Total 15 12 27 3 3 6 33

Seven embryogenic lines (250 mg fresh weight of embryogenic tissue per line) from five half-sib families were tested

Plant Cell Tiss Organ Cult

123

Discussion

The initiation of SE from megagametophytes containing

zygotic embryos of Stone pine was achieved at very low

frequency, with an overall mean around 0.5%. Although

the first papers that reported conifer SE in other pines, such

as P. strobus (Finer et al. 1989), pointed out that initiation

was not a limiting factor, the frequencies of initiation for

most species described in these early studies were also very

low (Becwar et al. 1990; Percy et al. 2000). To avoid

problems of genetic erosion in breeding programs, it is

desirable to capture as many genotypes as possible. For

pine species, new protocols are currently being developed

to improve initiation frequencies (Pullman and Johnson

2002), and therefore increased efficiency of SE in Stone

pine is expected. For example, with an optimized protocol,

the initiation frequency of P. strobus was increased to an

overall frequency of 76% (Klimaszewska et al. 2001).

Although most of the extrusions from the megagame-

tophyte tissue could be established as embryogenic lines in

some species (Miguel et al. 2004; Salajova and Salaj 2005),

differences between frequencies of extrusion and initiation

of SE were observed in other species (Percy et al. 2000;

Pullman and Johnson 2002). This poses a problem for

application of SE in some pine species in multivarietal

forestry, because it limits the number of captured geno-

types. However, extrusion is necessary for SE initiation. In

P. taeda, one-fifth of the extrusions led to initiation of

embryogenic lines (MacKay et al. 2006). In the case of

Stone pine, only less than 5% of extrusions in the 2005

experiment, and 8% in the 2006 experiment became

established as embryogenic lines. However, these impor-

tant differences between proportions of different years of

collection indicated that some factors control the viability

of extruded tissues. Therefore, their identification and

manipulation could improve the initiation of SE. In

P. taeda, EM extrusion and initiation were partly under

different types of genetic control (MacKay et al. 2006).

The presence of the female megagametophyte in the cul-

ture may be related to this effect, as discussed below.

Significant differences have been found in initiation

frequencies between collection years. These differences

have also been reported for other pine species, such as P.

pinaster (Miguel et al. 2004) and P. nigra (Salajova and

Salaj 2005). Our results clearly indicated that differences

between both years were maintained on all collection dates.

Differences in initiation of EM in P. taeda have been

ascribed to stimulatory compounds produced by the female

gametophyte (Pullman and Johnson 2002). Some authors

have also pointed out that observed maternal effects on the

initiation of embryogenic lines could be linked to the

presence of the megagametophyte in culture (MacKay

et al. 2006). Stone pine has one of the biggest

megagametophytes among conifers and, therefore, its

possible influence deserves to be studied.

Experiments on media composition in this study were

not broad enough to draw conclusions about their effect on

the initiation of embryogenic lines. However, it seems that

concentrations of 2,4-D and BAP, supplied within the

range commonly used in other pine species, were less

important than basal medium formulation. A similar

response was observed in P. pinaster, in which basal

medium was more important than 2,4-D concentrations in

the range of 2.4–13.5 lM (Park et al. 2006). However the

effect of 2,4-D concentrations could be influenced by the

endogenous content of regulators, which could explain the

observed differences between years. Although some stud-

ies reported that nutrient media composition was not a

determinant to achieve SE in some conifers, Stone pine

seems to belong to the group of species that are nutrient

media-dependent (Li et al. 1998; Miguel et al. 2004; Park

et al. 2006). Preliminary tests with several culture media

formulations commonly used for conifers, failed with

Stone pine. Using a nickel-containing mLV medium with

reduced cobalt, which improved initiation in recalcitrant

species like P. banksiana (Park et al. 2006), and modified

micronutrients led to positive responses in Stone pine. This

medium was superior to the standard mLV medium (Kli-

maszewska et al. 2001) when both were compared in the

2006 experiments. Although the presence of nickel and a

reduced level of cobalt and other micronutrients is proba-

bly beneficial, the presence of sucrose at different con-

centrations cannot be ignored. Therefore, more research is

needed to determine the role of medium components to

further improve initiation frequencies. However, it is

known that the mineral content and PGR supplement of

media used for initiation is not be the best medium for

maintaining embryo cleavage in proliferating EM. This

was also the case in Stone pine, in which the standard mLV

medium with 9.5 lM 2,4-D, 4.5 lM BAP and 20 g/l

sucrose, was the best medium for maintaining embryogenic

lines.

Selection of competent explants is critical for inducing

SE in conifers (Tautorus et al. 1991). The highest fre-

quencies of SE induction in Stone pine occurred when

zygotic embryos were in the post-cleavage polyembryony

to cotyledonary stages. These developmental stages are

common to other pine species, in which the precotyledo-

nary stage was the most responsive (Klimaszewska et al.

2001; Lelu et al. 1999; Salajova and Salaj 2005). It is

noteworthy that the developmental window for embryo-

genic response in Stone pine was almost identical to that

reported for P. pinaster, another Mediterranean pine spe-

cies (Miguel et al. 2004). In both species, maximal fre-

quencies of SE initiation occurred between the end of June

and the first week of July. Interestingly, the decrease in the

Plant Cell Tiss Organ Cult

123

percentage of established lines from 6 to 13 July, followed

by a slight recovery, which was observed for both years of

this study, was also reported for P. pinaster (Miguel et al.

2004).

It is well established that the several phases of plant

regeneration by SE are under genetic control (Park et al.

1993, 1994). Significant differences in somatic embryo-

genic response among families of conifers such as Picea

mariana (Cheliak and Klimaszewska 1991), Pinus strobus

(Garin et al. 1998; Klimaszewska et al. 2001), P. monticola

(Percy et al. 2000), P. pinaster (Miguel et al. 2004) P.

taeda (MacKay et al. 2006), and P. sylvestris (Lelu-Walter

et al. 2008; Niskanen et al. 2004) have been reported. In

Stone pine, significant differences for extrusion, and nearly

significant differences for initiation, were found among the

families. Some of the families performed better than others,

which suggested an additive genetic control on the initia-

tion of SE in Stone pine, as was reported in other conifers

(MacKay et al. 2006; Park et al. 1993). Differences in SE

initiation among mother trees were recorded in this study:

family 58 almost doubled the overall mean value, whereas

family 70 was five times below the mean value with regard

to frequency of initiation. These data are similar to those

obtained in a broader experiment with 20 half-sib families

of P. pinaster, in which the best family produced three

times the overall mean and the worst was six times below

the average (Miguel et al. 2004). Although frequencies of

initiation in Stone pine were very low, the fact that

embryogenic tissue was obtained from all the five families

tested is encouraging.

Although the number of families tested in this study was

relatively low, the difference in their ability to achieve

initiation showed that proper selection of mother trees

could be used to increase the number of captured geno-

types. This study did not detect any significant effect of

interaction between families and collection dates, as

reported in P. strobus (Klimaszewska et al. 2001). How-

ever, as was observed in that species, different temporal

patterns for initiation were recorded for the different fam-

ilies in the present study, likely reflecting developmental

disparities, as has been reported for other species (Lelu

et al. 1999), but also showing a family with a wider range

of responses than others. It is interesting that this particular

family also performed equally in both crop years. The

search for and selection of these ‘‘broad spectrum geno-

types’’ for initiation ability as maternal trees for controlled

crosses could be useful for increasing the number of cap-

tured genotypes in a breeding program. Selection of the

most favorable maternal parent has been proposed for

improving SE initiation (MacKay et al. 2006; Niskanen

et al. 2004).

The proliferation of EM in Stone pine was achieved by

continuous formation of proembryogenic and early

embryonic structures, which assumed either a smooth or

spiky morphotype, similar to that of another pine species

(Ramarosandratana et al. 2001a). The development of these

morphotypes seemed to be genotype dependent, although

sometimes changes within a given genotype in an uncon-

trolled manner were observed. Growth of EM was more

favorable with subculturing after dispersion on filter disks

than when using tissue clumps. The culture technique

based on dispersing embryogenic tissue by suspending it in

liquid medium and collecting it on filter paper disks, which

was originally developed to rescue slow-growing EM of

eastern white pine (Klimaszewska and Smith 1997), was

particularly useful for proliferating Stone pine embryo-

genic tissue. It grew faster than when it was cultured as

small tissue clumps, multiplying the initial ± 100 mg FW

by 52–96 times, depending on genotype, after 6-week

period. As this effect was marked in the case of the slower-

growing genotypes, in which growth was up to more 12

times higher when using the dispersal instead of the clump

procedure, the culture of initial extrusions following this

procedure will likely increase the number of established

lines (Lelu-Walter et al. 2008).

The main objective of the maturation experiments in this

study was to demonstrate that differentiation and mature

embryos could be obtained from established embryogenic

lines of Stone pine. In the present study, preculture on

medium with reduced nutrients improved the number of

mature embryos. Vigorous proliferation of embryogenic

tissue hampered differentiation and maturation of somatic

embryos, as in other conifers (Breton et al. 2006). There-

fore, slower growth caused by the reduction of nutrients in

the preconditioning medium might be behind the improved

results obtained in this study. In addition, coating

embryogenic tissue with AC clearly improved maturation,

as was also shown in P. pinaster (Lelu-Walter et al. 2006),

probably by adsorbing endogenously produced substances,

because it was more effective when the tissue was coated

than when it was added to the culture medium. Mature

embryos were obtained in five out of seven tested

embryogenic lines, which is around 70% of the tested

genotypes. Although the number of somatic embryos

obtained in this first study was very low, the percentage of

responsive genotypes is comparable, or even higher, than

in other pine species (Miguel et al. 2004). However,

research that involves aspects such as the medium’s water

status (Klimaszewska and Smith 1997; Ramarosandratana

et al. 2001b), carbon sources and their interaction with

gelling agents (Ramarosandratana et al. 2001a), anti-auxins

(Find et al. 2002), ethylene modulators (Kong and Yeung

1994), or other factors, may improve the efficiency of SE in

P. pinea.

Although the number of mature embryos obtained from the

maturation experiments described in this study was too low to

Plant Cell Tiss Organ Cult

123

draw any conclusion about their germination and conversion

abilities, the high percentages of germination (70%) and

conversion (30%) that were obtained are encouraging.

Acknowledgments The authors gratefully thank N. Cleto and Y.

Vinuesa for their technical assistance. Funds were provided by pro-

jects AGL2002-00867 and AGL2005-07585, and IMIDRA and INIA

grants to E. Carneros. We wish to thank the National Forest Breeding

Centre ‘‘Puerta de Hierro’’ (Madrid) of the Spanish Ministry of

Environment, and Dr. Mutke for all their help in collecting plant

material. We thank the Canadian Forest Service for hosting E.

Carneros at its laboratory in Fredericton.

References

Becwar MR, Nagmani R, Wann SR (1990) Initiation of embryogenic

cultures and somatic embryo development in loblolly pine

(Pinus taeda). Can J Res 20:810–817. doi:10.1139/x90-107

Bonga JM (2004) The effect of various culture media on the formation

of embryo-like structures in cultures derived from explants taken

from mature Larix decidua. Plant Cell Tissue Organ Cult 77:43–

48. doi:10.1023/B:TICU.0000016488.79965.b7

Breton D, Harvengt L, Trontin J-F et al (2006) Long-term subculture

randomly affects morphology and subsequent maturation of

early somatic embryos in maritime pine. Plant Cell Tissue Organ

Cult 87:95–108. doi:10.1007/s11240-006-9144-9

Capuana M, Giannini R (1995) In vitro plantlet regeneration from

embryonic explants of Pinus pinea L. In Vitro Cell Dev Biol

Plant 31:202–206. doi:10.1007/BF02632022

Cheliak WM, Klimaszewska K (1991) Genetic variation in somatic

embryogenic response in open-pollinated families of black spruce.

Theor Appl Genet 82:185–190. doi:10.1007/BF00226211

Deb CR, Tandon P (2002) Somatic embryogenesis and plantlet

regeneration from mature zygotic embryos of Pinus kesiya (Royle

ex. Gord.). J Plant Biol 29:301–306

Diamantoglou S, Panagopoulos I, Munoz-Ferriz A et al (1990) In vitro

studies of embryo growth, callus formation and multiple bud

induction of Pinus pinea L. J Plant Physiol 137:58–63

Duchefa Biochemie BV (2003) Biochemicals, Plant cell and tissue

culture, Plant molecular biochemicals. Catalogue 2003–2005.

Haarlem, The Netherlands

El-Kassaby Y (2004) Feasibility and proposed outline of a global

review of forest biotechnology. Forest Genetic Resources Work-

ing Paper FGR/77E: Forest Resources Development Service,

Forest Resources Division. FAO, Rome

Find J, Grace L, Krogstrup P (2002) Effect of anti-auxins on maturation

of embryogenic tissue cultures of Nordmanns fir (Abies nordman-niana). Physiol Plant 116:231–237. doi:10.1034/j.1399-3054.

2002.1160213.x

Finer JJ, Kriebel HB, Becwar MR (1989) Initiation of embryogenic

callus and suspension cultures of eastern white pine (Pinusstrobus L). Plant Cell Rep 8:203–206. doi:10.1007/BF00778532

Garcıa-Ferriz L, Serrano L, Pardos JA (1994) In vitro shoot organo-

genesis from excised immature cotyledons and microcuttings

production in Stone Pine. Plant Cell Tissue Organ Cult 36:135–

140. doi:10.1007/BF00048324

Garin E, Isabel N, Plourde A (1998) Screening of large numbers of

seed families of Pinus strobus L. for somatic embryogenesis

from immature and mature zygotic embryos. Plant Cell Rep 18:

37–43. doi:10.1007/s002990050528

Gonzalez MV, Rey M, Tavazza R et al (1998) In vitro adventitious

shoot formation on cotyledons of Pinus pinea. HortScience 33:

749–750

Gupta PK, Grob JA (1995) Somatic embryogenesis in conifers. In:

Jain S, Gupta PK, Newton RJ (eds) Somatic embryogenesis in

woody plants, vol 1. Kluwer Academic, Dordrecht, pp 81–98

Klimaszewska K, Cyr DR (2002) Conifer somatic embryogenesis: I.

Development. Dendrobiology 48:31–39

Klimaszewska K, Smith D (1997) Maturation of somatic embryos of

Pinus strobus is promoted by a high concentration of gellan gum.

Physiol Plant 100:949–957. doi:10.1111/j.1399-3054.1997.tb00

022.x

Klimaszewska K, Park Y-S, Overton C et al (2001) Optimized

somatic embryogenesis in Pinus strobus L. In Vitro Cell Dev

Biol Plant 37:392–399. doi:10.1007/s11627-001-0069-z

Kong L, Yeung EC (1994) Effects of ethylene and ethylene inhibitors

on white spruce somatic embryo maturation. Plant Sci 104:71–80.

doi:10.1016/0168-9452(94)90192-9

Lelu MA, Bastien C, Drugeault A et al (1999) Somatic embryogen-

esis and plantlet development in Pinus sylvestris and Pinuspinaster on medium with and without growth regulators. Physiol

Plant 105:719–728. doi:10.1034/j.1399-3054.1999.105417.x

Lelu-Walter MA, Bernier-Cardou M, Klimaszewska K (2006)

Simplified and improved somatic embryogenesis for clonal

propagation of Pinus pinaster (Ait). Plant Cell Rep 25:767–776.

doi:10.1007/s00299-006-0115-8

Lelu-Walter MA, Bernier-Cardou M, Klimaszewska K (2008) Clonal

plant production from self- and cross-pollinated seed families of

Pinus sylvestris (L.) through somatic embryogenesis. Plant Cell

Tissue Organ Cult 92:31–45. doi:10.1007/s11240-007-9300-x

Li X, Huang F, Gbur E (1998) Effect of basal medium, growth

regulators and phytagel concentration of embryogenic cultures

from immature zygotic embryos of loblolly pine (Pinus taeda L.).

Plant Cell Rep 17:298–301. doi:10.1007/s002990050396

Litvay JD, Verma DC, Johnson MA (1985) Influence of loblolly pine

(Pinus taeda L.). Culture medium and its components on growth

and somatic embryogenesis of the wild carrot (Daucus carotaL.). Plant Cell Rep 4:325–328. doi:10.1007/BF00269890

MacKay JJ, Becwar MR, Park Y-S et al (2006) Genetic control of

somatic embryogenesis initiation in loblolly pine and implications

for breeding. Tree Genet Genomes 2:1–9. doi:10.1007/s11295-

005-0020-2

Merkle SA, Nairn CJ (2005) Hardwood tree biotechnology. In Vitro

Cell Dev Biol Plant 41:602–619. doi:10.1079/IVP2005687

Miguel C, Goncalves S, Tereso S et al (2004) Somatic embryogenesis from

20 open-pollinated families of Portuguese plus trees of maritime pine.

Plant Cell Tissue Organ Cult 76:121–130. doi:10.1023/B:TICU.

0000007253.91771.e3

Mutke S, Gordo J, Gil L (2000) The stone pine (Pinus pinea L.)

breeding programme in Castile-Leon (Central Spain). FAO-

CIHEAM NUCIS-Newsletter 9:50–55

Nehra NS, Becwar MR, Rottman WH et al (2005) Forest biotech-

nology: Innovative methods, emerging opportunities. In Vitro

Cell Dev Biol Plant 41:701–717. doi:10.1079/IVP2005691

Niskanen AM, Lu J, Seitz S et al (2004) Effect of parent genotype on

somatic embryogenesis in Scots pine (Pinus sylvestris). Tree

Physiol 24:1259–1265

Oliveira P, Barriga J, Cavaleiro C et al (2003) Sustained in vitro root

development obtained in Pinus pinea L inoculated with ectomy-

corrhizal fungi. Forestry 76:579–587. doi:10.1093/forestry/76.5.

579

Park Y-S (2002) Implementation of conifer somatic embryogenesis in

clonal forestry: technical requirements and deployment consid-

erations. Ann Sci 59:651–656. doi:10.1051/forest:2002051

Park Y-S, Pond SE, Bonga JM (1993) Initiation of somatic

embryogenesis in white spruce (Picea glauca): genetic control,

culture treatment effects, and implications for tree breeding.

Theor Appl Genet 86:427–436. doi:10.1007/BF00838557

Plant Cell Tiss Organ Cult

123

Park Y-S, Pond SE, Bonga JM (1994) Somatic embryogenesis in

white spruce (Picea glauca): genetic control in somatic embryos

exposed to storage, maturation treatments, germination, and

cryopreservation. Theor Appl Genet 89:742–750. doi:10.1007/

BF00223714

Park Y-S, Lelu-Walter MA, Harvengt L et al (2006) Initiation of

somatic embryogenesis in Pinus banksiana, P. strobus, P.

pinaster and P. sylvestris at three laboratories in Canada and

France. Plant Cell Tissue Organ Cult 86:87–101. doi:10.1007/

s11240-006-9101-7

Percy RE, Klimaszewska K, Cyr DR (2000) Evaluation of somatic

embryogenesis for clonal propagation of western white pine. Can

J Res 30:1867–1876. doi:10.1139/cjfr-30-12-1867

Pullman GS, Johnson S (2002) Somatic embryogenesis in loblolly

pine (Pinus taeda L.): improving culture initiation rates. Ann Sci

59:663–668. doi:10.1051/forest:2002053

Radojevic L, Alvarez C, Fraga MF et al (1999) Somatic embryogenic

tissue establishment from mature Pinus nigra Arn. Ssp.

Salzmannii embryos. In Vitro Cell Dev Biol Plant 35:206–209.

doi:10.1007/s11627-999-0078-x

Ramarosandratana A, Harvengt L, Bouvet A et al (2001a) Influence

of the embryonal-suspensor mass (ESM) sampling on develop-

ment and proliferation of maritime pine somatic embryos. Plant

Sci 160:473–479. doi:10.1016/S0168-9452(00)00410-6

Ramarosandratana A, Harvengt L, Bouvet A et al (2001b) Effects

of carbohydrate source, polyethylene glycol and gellan gum

concentration on embryonal-suspensor mass (ESM) proliferation

and maturation of maritime pine somatic embryos. In Vitro Cell Dev

Biol Plant 37:29–34. doi:10.1007/s11627-001-0006-1

Salajova T, Salaj J (2005) Somatic embryogenesis in Pinus nigra:

embryogenic tissue initiation, maturation and regeneration ability

of established cell lines. Biol Plant 49:333–339. doi:10.1007/

s10535-005-0003-z

Tang W, Guo Z, Ouyang F (2001) Plant regeneration from

embryogenic cultures initiated from mature loblolly pine zygotic

embryos. In Vitro Cell Dev Biol Plant 37:558–563. doi:10.1007/

s11627-001-0097-8

Tautorus TE, Fowke L, Dunstan DI (1991) Somatic embryogenesis in

conifers. Can J Bot 69:1873–1899. doi:10.1139/b91-237

Valdes AE, Ordas RJ, Fernandez B et al (2001) Relationships

between hormonal contents and the organogenic response in

Pinus pinea cotyledons. Plant Physiol Biochem 39:377–384. doi:

10.1016/S0981-9428(01)01253-0

von Arnold S, Sabala I, Bozhkov P et al (2002) Developmental

pathways of somatic embryogenesis. Plant Cell Tissue Organ Cult

69:233–249. doi:10.1023/A:1015673200621

Zoglauer K, Behrendt U, Rahmat A (2003) Somatic embryogenesis—

the gate to biotechnology in conifers. In: Laimer M, Rucker W et al

(eds) Plant tissue culture—100 years since Gottlieb Haberlandt.

Springer, Heidelberg, pp 175–202

Plant Cell Tiss Organ Cult

123