ORIGINAL PAPER

Patterning during somatic embryogenesis in Scots pine in relationto polar auxin transport and programmed cell death

Malin Abrahamsson • Silvia Valladares •

Emma Larsson • David Clapham • Sara von Arnold

Received: 23 September 2011 / Accepted: 19 December 2011 / Published online: 30 December 2011

� Springer Science+Business Media B.V. 2011

Abstract Somatic embryogenesis is a useful tool to

propagate conifers vegetatively. However, a major limita-

tion in many pine species is the low quality of cotyledonary

somatic embryos. The aim of this study has been to elu-

cidate the developmental pathway of somatic embryos in

Scots pine (Pinus sylvestris), to identify deviations from

the normal pathway and to identify processes that might

disturb normal development. Initially we compared the

developmental pathway of somatic embryogenesis in rep-

resentative cell lines yielding cotyledonary embryos with

normal and abnormal morphology. Early embryos carrying

suspensor cells in excess of the normal number (supernu-

merary) were more frequent in cell lines giving rise to

abnormal cotyledonary embryos. In this study we show that

the frequency of early somatic embryos with supernumer-

ary suspensor cells increased after treatment with the auxin

transport inhibitor 1-N-naphtylphthalamic acid (NPA).

Furthermore, the yield of developing embryos increased

significantly after treatment with the antiauxin 2-(4-chlo-

rophenoxy)-2-methylpropionic acid (PCIB), but the mor-

phology of the embryos was not affected. The number of

cells undergoing PCD was analyzed using a TUNEL-assay.

The frequency of TUNEL-positive cells was high both in

proliferating cultures and during differentiation of early

somatic embryos. However, the pattern of TUNEL-positive

cells was similar in normal somatic embryos and in

embryos with supernumerary suspensor cells. Together our

results suggest that the presence of supernumerary sus-

pensor cells in early somatic embryos of Scots pine is

caused by disturbed polar auxin transport and results in

aberrant embryo development.

Keywords Embryo patterning � NPA � PCIB � Pinus

sylvestris � Polar auxin transport � Programmed cell death �Scots pine � Somatic embryogenesis

Abbreviations

2,4-D 2,4-dichlorophenoxyacetic acid

ABA Abscisic acid

BA N6-benzyladenine

DCR Basal culture medium (Gupta and Durzan 1985)

DMSO Dimethyl sulfoxide

IAA Indoleacetic acid

NPA 1-N-napthylphthalamic acid

PAT Polar auxin transport

PCD Programmed cell death

PCIB 2-(4-chlorophenoxy)-2-methylpropionic acid

PEG Polyethylene glycol

PEM Proembryogenic mass

PGR Plant growth regulator (auxin and cytokinin)

TUNEL Terminal deoxynucleotidyl transferase (TdT)-

mediated dUTP nick end labelling

Malin Abrahamsson and Silvia Valladares contributed equally to this

work.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11240-011-0103-8) contains supplementarymaterial, which is available to authorized users.

M. Abrahamsson (&) � S. Valladares � E. Larsson �D. Clapham � S. von Arnold

Department of Plant Biology and Forest Genetics, Uppsala

BioCenter, Swedish University of Agricultural Sciences,

P.O. Box 7080, 750 07 Uppsala, Sweden

e-mail: Malin.Abrahamsson@slu.se

Present Address:S. Valladares

Department of Plant Physiology, Instituto de Investigaciones

Agrobiologicas de Galicia (CSIC), Apartado 122,

15780 Santiago de Compostela, Spain

123

Plant Cell Tiss Organ Cult (2012) 109:391–400

DOI 10.1007/s11240-011-0103-8

Introduction

Somatic embryogenesis is an attractive method to propa-

gate conifers vegetatively and is employed both as a tool in

the breeding program and as a method for large scale clonal

propagation. The whole process of regeneration of conifer

plants through somatic embryogenesis comprises a

sequence of steps: initiation and proliferation of embryo-

genic cultures, differentiation of early embryos and

development of late and cotyledonary embryos (von

Arnold and Clapham 2008). Many conifers belonging to

the genus spruce (Picea) can be propagated on a large scale

by somatic embryos (Klimaszewska et al. 2007). However,

for several species belonging to the genus Pinus the initi-

ation frequency of embryogenic cell lines is low and

regeneration of high quality cotyledonary embryos is poor

(Pullman et al. 2008; Bonga et al. 2010).

Development of multiple embryos from a single zygote

by monozygotic polyembryony is common among gym-

nosperms, including pines, but is rare in spruces (Singh

1978). During polyembryony the early embryo goes

through several rounds of cleavage resulting in many

equal-sized embryos. These embryos start to compete for

survival, and ultimately one embryo becomes dominant,

while the subordinate embryos are aborted (Nagmani et al.

1995; Filonova et al. 2002). In most pine species, initiation

of embryogenic cultures is limited to the first weeks of

zygotic embryo development during the cleavage stage

(Keinonen-Mettala et al. 1996; Haggman et al. 1999; Lelu-

Walter et al. 2008) or prior to the emergence of cotyle-

donary embryos (Becwar et al. 1990; Lelu et al. 1999). It

has been suggested that embryogenic cultures initiated

from immature zygotic embryos during the cleavage stage

result from a continuation of the cleavage process (Bozh-

kov et al. 1997; Park et al. 2006). In contrast, embryogenic

cultures of spruces are usually initiated from differentiated

cells in mature zygotic embryos, after these cells have been

stimulated to dedifferentiate (Mo et al. 1996).

In order to efficiently regulate the formation of plants

via somatic embryogenesis, we need to understand how

somatic embryos develop. Ideally, such knowledge should

be gained through the construction of a fate map repre-

senting an adequate number of morphological and molec-

ular markers specifying distinct developmental stages.

Pullman et al. (2003) developed a staging system in lob-

lolly pine (Pinus taeda) to evaluate the morphological

development in zygotic and somatic embryos. However,

our knowledge about how somatic embryos in pine species

develop is still insufficient to allow detailed descriptive

characterization. In contrast, somatic embryogenesis in

Norway spruce (Picea abies) has been thoroughly studied.

A time-lapse tracking technique was used to analyse the

developmental pathway of somatic embryos (Filonova

et al. 2000a). Based on this knowledge the whole process

can be synchronized and controlled (von Arnold and

Clapham 2008).

Auxin plays an essential role in plant development.

Critical to its activity as a developmental regulator is its

polar intercellular transport. Embryo patterning mutants in

Arabidopsis have been used for studying polarization

during early embryo development (Friml et al. 2003).

Auxin-regulated pattern formation has also been studied by

treating embryos with antiauxins such as 2-(4-chloro-

phenoxy)-2-methylpropionic acid (PCIB) or auxin trans-

port inhibitors such as 1-N-napthylphthalamic acid (NPA).

PCIB treatment of embryogenic cultures in different

coniferous species results in reduced proliferation and

increased quality of the mature embryos (Find et al. 2002;

Liao et al. 2008). NPA-treated somatic embryos of Norway

spruce attain an aberrant morphology as a consequence of

blocked polar auxin transport (PAT) (Larsson et al. 2008a).

PAT is crucial for appropriate apical-basal patterning

during early embryogeny in Norway spruce and polariza-

tion proceeds through the establishment of three major cell

types: the meristematic cells of the embryonal mass, the

embryonal tube cells and the terminally differentiated

suspensor cells. The suspensor cells in early somatic

embryos of Norway spruce are eliminated by programmed

cell death (PCD) (Filonova et al. 2000a, b).

The aim of this study was to elucidate the development

of somatic embryos in Scots pine, to identify deviations

from normal development and to identify processes that

might cause the abnormal development. We initially ana-

lyzed the developmental pattern of somatic embryos in cell

lines of Scots pine giving rise to cotyledonary embryos

with normal morphology, or to cotyledonary embryos with

abnormal morphology which do not develop into plants.

During differentiation of early embryos, the formation of

supernumerary suspensor cells, that is cells in excess of the

normal number, was more common in cell lines giving rise

to embryos with abnormal morphology. We treated cul-

tures with PCIB or NPA to examine the effects of reduced

auxin activity or inhibited polar transport on the occurrence

of supernumerary suspensor cells and aberrant embryo

development. Furthermore, the proportion of cells going

through PCD was analyzed using TUNEL-assay.

Materials and methods

Plant material

Embryogenic cell lines of Scots pine (Pinus sylvestris L.)

were initiated in 2000 from immature zygotic embryos

collected from open-pollinated trees growing in a seed

orchard in central Sweden (Burg et al. 2007). The cell lines

392 Plant Cell Tiss Organ Cult (2012) 109:391–400

123

were stored in liquid nitrogen and thawed before starting

the experiments. Three embryogenic cell lines were used in

this study: cell line 12:12 which produces cotyledonary

embryos with normal morphology (Fig. 1a) but also some

with abnormal morphology (Fig. 1b) and cell lines 3:6 and

3:10 which give rise to abnormal cotyledonary embryos

(Fig. 1c, d).

DCR medium (Gupta and Durzan 1985) modified as

described previously (Burg et al. 2007) was used as basal

medium. The cultures were proliferated on DCR prolifer-

ation medium, supplemented with 2,4-dichlorophenoxy-

acetic acid (2,4-D) and N6-benzyladenine (BA) at 9.0 and

4.4 lM, respectively, as plant growth regulators (PGRs)

and 1% sucrose. To stimulate embryo maturation, the

cultures were first pretreated on PGR-free DCR medium,

pre-maturation medium, for 2–3 weeks and then trans-

ferred to DCR maturation medium, containing 7.5%

polyethylene glycol (PEG 4000), 60 lM abscisic acid

(ABA), 3% maltose and 0.35% gellan gum (Gelrite�,

Kelco) for 8–11 weeks. The cultures were incubated in

darkness at 22 ± 1�C and sub-cultured every second to

third week.

Histological preparations

Mature embryos from cell line 12:12 were collected for

sectioning after 10 weeks on maturation medium. The

embryos were fixed, embedded and serial-sectioned as

earlier described by Filonova et al. (2000a).

Cell tracking

Cell-tracking experiments were performed with cell line

3:6, 3:10 and 12:12 in thin layers of agarose (Filonova et al.

2000a). For analyzing the developmental pattern, about 50

aggregates per cell line were preselected at the start of the

maturation treatment. Their development was documented

every 3rd to 4th day, over 25 days. The development of

somatic embryos was separated into different stages based

on the developmental pathway of somatic embryos in cell

line 12:12 (Fig. 2).

Antiauxin treatment

Embryogenic cultures from cell lines 3:10 and 12:12 were

exposed to 2-(4-chlorophenoxy)-2-methylpropionic acid

(PCIB) (Sigma-Aldrich) during the pre-maturation treat-

ment. PCIB was dissolved in 95% (v/v) ethanol and added

to the medium after autoclaving at different concentrations

(1–20 lM). The strongest effect was observed at 10 lM

PCIB, while at higher concentrations the survival rate

decreased (data not shown). Therefore, we present data

only from cultures treated with 10 lM PCIB. After the

PCIB treatment the cultures were transferred to maturation

medium.

About 500 mg (f.w.) tissue was transferred to each Petri

dish (diameter 60 mm) and each PCIB treatment included

ten replicates. Differentiation of early somatic embryos

(Fig. 2d) was analyzed directly after 2 weeks on pre-mat-

uration medium. The samples were re-suspended in dH20

and poured into Petri dishes for microscopic examination.

The yield of early somatic embryos differentiated from 100

Fig. 1 Fully matured cotyledonary embryos. a Embryo with 6

developed cotyledons and elongated hypocotyl from cell line 12:12.

b Embryo with 3 developed cotyledons and elongated hypocotyl from

cell line 12:12. c Embryo with 6 developed cotyledons and aborted

hypocotyl from cell line 3:10. d Embryo with 5 developed cotyledons

and without distinct hypocotyl from cell line 3:6. (e and f) Longitu-

dinal sections of embryos from cell line 12.12. (e) Embryo with 5

developed cotyledons and shoot meristem. (f) Embryo with 2

cotyledons and aborted shoot meristem. co cotyledon, hc hypocotyl,

sm shoot meristem. Bars 250 lm

Plant Cell Tiss Organ Cult (2012) 109:391–400 393

123

aggregates was determined in three replicates per treat-

ment. The yield and morphology of mature somatic

embryos (Fig. 2j) were recorded in all ten replicates after

8 weeks on maturation medium. The data were analyzed

for significance using ANOVA (analysis of variance,

P B 0.05). All data analysis in these studies was performed

using the SPSS� v. 17.0 statistical software package.

Inhibition of polar auxin transport

The effect of polar auxin transport (PAT) on the differen-

tiation of somatic embryos was assessed by exposing

embryogenic cultures of cell line 3:10 and 12:12 to the

auxin transport inhibitor 1-N-napthylphthalamic acid

(NPA) (Sigma-Aldrich). NPA was dissolved in dimethyl

sulfoxide (DMSO) to obtain a stock solution of 100 mM.

The cultures were treated with 20 and 30 lM NPA during

2 weeks on pre-maturation medium and 2–3 weeks on

maturation medium. The clearest effect was observed after

treatments with 30 lM NPA, therefore we present data

only from this treatment. Media lacking additives as well

as media supplemented with DMSO were used as controls.

Since no effect of DMSO was observed we have not

included data from DMSO controls.

The amount of tissue transferred to each Petri dish

(diameter 60 mm) at the start of the experiments was kept

constant at approximately 500 mg (f.w.). Each NPA

treatment included ten replicates. Differentiation of early

somatic embryos (Fig. 2d) was assessed after 2 weeks on

maturation medium. The samples were re-suspended in

dH20 and poured into Petri dishes for microscopic exami-

nation. The morphology of early somatic embryos differ-

entiated from 200 aggregates was determined in three

replicates per treatment. The yield and the morphology of

the mature somatic embryos (Fig. 2j) were recorded in all

ten replicates after 10–11 weeks on maturation medium.

The data were analyzed for significance using ANOVA

(analysis of variance, P \ 0.1 or P \ 0.05).

In situ detection of DNA fragmentation (TUNEL assay)

The number of cells going through PCD in embryogenic

cultures of Norway spruce has successfully been studied by

using terminal deoxynucleotidyl transferase (TdT)-mediated

Fig. 2 Development of somatic embryos of Scots pine in cell line

12:12. a Proliferating embryogenic cell aggregate in the presence of

plant growth regulators (PGRs) (stage 1a). b Slightly globular

structure with densely packed cells covered by a smooth surface

2–3 weeks after withdrawal of PGRs (stage 1b). c Early embryo after

1–2 weeks exposure to abscisic acid (ABA) (stage 1c). d Early

somatic embryo after 1–3 weeks exposure to ABA (stage 2). e Late

embryo after 2–5 weeks exposure to ABA (stage 3). f Late embryo

before cotyledon differentiation after 5–6 weeks exposure to ABA

(stage 4). g–i Maturing embryos with developing cotyledonary

primordia after 6–8 weeks exposure to ABA (stages 5–7). j Fully

matured cotyledonary embryo after 10 weeks exposure to ABA (stage

8). co cotyledon, em embryonal mass, hc hypocotyl, mc meristematic

cells, s suspensor, vc vacuolated cells. Bars, 100 lm (a–e); 250 lm

(f–j)

394 Plant Cell Tiss Organ Cult (2012) 109:391–400

123

dUTP nick end labelling (TUNEL), which is an in situ

method to label fragmented DNA (Filonova et al. 2000b).

Samples for in situ detection of DNA fragmentation were

collected from cell line 3:10 and 12:12: after 2 weeks on

proliferation medium, after 2 weeks on pre-maturation

medium and after 2 weeks on maturation medium. Nuclear

DNA fragmentation was assessed by whole mount TUNEL

(In situ Cell Death Detection Kit, Roche, Penzberg,

Germany) as described earlier by Larsson et al. (2008a). The

frequency of TUNEL-positive cells was based on the total

number of DAPI-stained nuclei in each structure. In total, 15

randomly chosen structures from each sample were ana-

lyzed. The data were analyzed for significance using t test,

P B 0.05.

In order to assess if PCD is suppressed in embryos

carrying supernumerary suspensor cells, TUNEL assays

were performed on somatic embryos at stage 1c, 2, 3 and 4

(Fig. 2c, d, e, f) collected after 2 and 4 weeks on matura-

tion medium. In total 52 embryos from cell line 3:10 and

cell line 12:12 were analyzed.

Results and discussion

Time-lapse tracking

Embryogenic cell lines of Scots pine vary significantly in

their ability to differentiate cotyledonary embryos (Burg

et al. 2007). Furthermore, the morphology of the cotyle-

donary embryos differs among cell lines. Initially we

analyzed the developmental pathway of somatic embryo-

genesis in eight cell lines, of which three representative cell

lines were chosen for more detailed analysis: cell line

12:12 which produces predominantly cotyledonary

embryos with normal morphology (Fig. 1a) and cell lines

3:6 and 3:10 which give rise to abnormal cotyledonary

embryos (Fig. 1c, d).

The development of somatic embryos was separated into

different stages (stage 1–8, Fig. 2). Proliferating embryo-

genic cultures in cell line 12:12 consisted of a mixture of

small meristematic cells and elongated vacuolated cells,

resembling the proembryogenic masses (PEMs) in Norway

spruce (Filonova et al. 2000a). Most of the cells were

clustered into large aggregates, stage 1a (Fig. 2a). At the

periphery of the large aggregates some regions ‘budded

off’, giving rise to new separate aggregates which

increased in size. After withdrawal of PGRs the cultures

continued to proliferate by forming new aggregates at the

same time as parts of the clustered aggregates became more

globular. These globular regions consisted of densely

packed cells covered by a smooth surface, stage 1b

(Fig. 2b). The cultures continued to proliferate after

transfer to maturation medium simultaneously as new

globular structures in the clustered aggregates were formed

and early somatic embryos started to differentiate, stage 1c

(Fig. 2c). Subsequently, early (stage 2, Fig. 2d) and late

(stage 3, Fig. 2e) somatic embryos composed of an

embryonal mass and a suspensor developed. The suspensor

cells started to degrade during stage 4 (Fig. 2f) and were

eliminated around stage 7 and 8 (Fig. 2i, j). The hypocotyl

started to elongate at stage 4 and the differentiation of the

cotyledons along with further elongation of the hypocotyl

took place at stage 5 (Fig. 2g). The number of cotyledons

varied from two to six and about 50% of the somatic

embryos carried four or more cotyledons and most of them

had a shoot meristem (Fig. 1e). Somatic embryos with two

cotyledons lacked a shoot meristem (Fig. 1f).

In cell line 3:6 and 3:10 the somatic embryos usually

started to differentiate after 1 week, and a high proportion

of both early and late somatic embryos carried supernu-

merary suspensor cells, resulting in an unbalanced ratio

between the embryonal mass and the suspensor (Fig. 4c).

Furthermore, the cotyledons started to differentiate already

by stage 4 (Fig. 2f). The number of cotyledons varied from

two to six and about one-third of the embryos carried four

or more cotyledons. The somatic embryos showed radial

growth during the whole process of maturation, resulting in

a high frequency of embryos with aborted or abnormal

hypocotyl (Fig. 1c, d).

There were no fundamental differences between the cell

lines in the developmental pathway leading to cotyledonary

embryos. However, already at the stage of early embry-

ogeny a significantly higher frequency of the somatic

embryos in cell line 3:6 and 3:10 carried supernumerary

suspensor cells compared to in cell line 12:12. About 35%

of the early somatic embryos in cell line 3:10 and only 8%

of the embryos in cell line 12:12 carried supernumerary

suspensor cells. The developmental programs of the

embryo proper and the suspensor are closely coordinated,

and imbalance causes embryo defects and lethality

(Smertenko et al. 2003; Bozhkov et al. 2005).

Decreased endogenous auxin activity stimulates

differentiation of early somatic embryos

In several plant species it has been shown that the endog-

enous indoleacetic acid (IAA) level must be kept low to

ensure normal somatic embryo formation (Korlach and

Zoglaur 1995; Find et al. 2002). It has been proposed that

the auxin antagonist PCIB reduces the activity of endoge-

nous IAA by competitive binding to auxin receptors

(McRae and Bonner 1953). In order to determine whether a

decreased auxin activity could reduce the proliferation of

embryogenic tissue of Scots pine during the pre-maturation

and maturation treatments and promote normal embryo

development, embryogenic cultures of cell line 3:10 and

Plant Cell Tiss Organ Cult (2012) 109:391–400 395

123

12:12 were treated with 10 lM PCIB during pre-matura-

tion. In both cell lines the treatment reduced proliferation

of embryogenic cultures on pre-maturation and maturation

media. However, we could not observe any changes in the

morphology of early somatic embryos after treatment with

PCIB. The yield of cotyledonary embryos was significantly

increased after the PCIB treatment (Fig. 3). In both control

cotyledonary somatic embryos and in PCIB-treated

embryos about 33% of the cotyledonary embryos in cell

line 3:10 carried four or more cotyledons, and in cell line

12:12 about 50% of the cotyledonary embryos carried four

or more cotyledons. Therefore, in contrast to what has been

reported previously for other conifers (Find et al. 2002;

Liao et al. 2008), reduced auxin activity does not improve

the morphology either in developing embryos or in coty-

ledonary embryos of Scots pine. This indicates that the

formation of supernumerary suspensor cells is not caused

by too high endogenous auxin content. We cannot, how-

ever, exclude that auxin homeostasis is disturbed in

embryogenic cultures of Scots pine, since the yield of

developing embryos increased.

Inhibition of PAT results in abnormal embryo

morphology

NPA-treatment during development of somatic embryos in

Norway spruce led to an increased endogenous IAA con-

tent, abnormal cell divisions and decreased PCD, resulting

in a changed balance between the embryonal mass and the

suspensor (Larsson et al. 2008a). In order to test if dis-

turbed PAT can be one of the reasons for the formation of

supernumerary suspensor cells in Scots pine somatic

embryos, we analyzed how embryo morphology was

affected when the cultures were treated with an auxin

transport inhibitor.

Early somatic embryos, after 2 weeks on maturation

medium, could be separated into four categories based on

their morphology (Fig. 4). Under standard culture condi-

tions a high proportion of the embryos had a normal

morphology (Table 1). The most striking difference in

embryo morphology between cell line 3:10 and 12:12 was

the higher proportion of embryos with supernumerary

suspensor cells in cell line 3:10. The total yield of differ-

entiating somatic embryos increased significantly when the

cultures were treated with 30 lM NPA during 2 weeks on

pre-maturation medium and 2 weeks on maturation med-

ium (Table 1). A similar increase in the yield of somatic

embryos after NPA treatment was shown in Norway spruce

(Larsson et al. 2008a). In both cell lines of Scots pine the

proportion of normal somatic embryos decreased after

NPA treatment (Table 1). In cell line 12:12 the proportion

of somatic embryos with supernumerary suspensor cells

increased, suggesting that this phenotype is caused by

disturbed PAT. In contrast, in cell line 3:10 the proportion

of embryos with supernumerary suspensor cells decreased

after NPA treatment. The higher proportion of embryos

with supernumerary suspensor cells in control cultures in

Fig. 3 Treatment with the anti-auxin PCIB stimulates embryo

differentiation. Embryogenic cultures of cell lines 12:12 (grey bars)

and 3:10 (black bars) were treated with 10 lM PCIB during the pre-

maturation phase. The average number of embryos at stage 7 (Fig. 2i)

per gram of embryogenic tissue was determined after 8 weeks on

maturation medium. The data are based on ten replicates. Within each

cell line an asterisk denotes significant differences between the

control and PCIB treatment at the P B 0.05 level according to

Duncan’s multiple range test

Fig. 4 Different morphologies of early somatic embryos of cell line

12:12. a Embryo with a normal morphology, b Twin embryo with

partially fused embryonal masses, c Embryo with supernumerary

suspensor cells, d Degenerating embryo with vacuolated cells in the

embryonal mass and meristematic cells in the suspensor. emembryonal mass, s suspensor; vc vacuolated cells. Bars, 100 lm

396 Plant Cell Tiss Organ Cult (2012) 109:391–400

123

cell line 3:10 than in cell line 12:12 indicates that PAT is

less efficient in somatic embryos in cell line 3:10 and that

further reduction of PAT by treatment with NPA leads to

embryo degeneration. We suggest that the presence of

supernumerary suspensor cells in early somatic embryos of

Scots pine is caused by disturbed PAT. In accordance, in

Norway spruce embryos, blocked PAT during differentia-

tion of the suspensor stimulates cell division of the meri-

stematic cells adjacent to the tube cells and this causes the

formation of supernumerary suspensor cells (Larsson et al.

2008b).

In both cell lines the proportion of somatic embryos with

separated cotyledons decreased and embryos carrying

partially or severely fused cotyledons increased after

treatment with NPA (Supplementary Fig. 1). The propor-

tion of embryos with partially and severely fused cotyle-

dons after NPA treatment was higher in cell line 3:10, but,

owing to a high variation among replicates, the differences

between the cell lines were not significant. The aberrant

morphologies of cotyledonary somatic embryos after

treatment with NPA are comparable with what has previ-

ously been shown in Norway spruce (Larsson et al. 2008a;

Hakman et al. 2009). In Norway spruce, normal cotyle-

donary somatic embryos were formed when the cultures

were exposed to NPA only during the earlier stages, while

embryos treated with NPA during development of late

embryos displayed apical abnormalities including fused or

aborted cotyledons (Larsson et al. 2008a). Based on the

fact that the proportion of Scots pine control embryos with

severely fused cotyledons was low we assume that PAT has

been activated during development of late embryos.

Suspensor cells are degraded by PCD

The frequency of TUNEL-positive cells is much higher in

proliferating embryogenic cultures of Scots pine than in

Norway spruce, 25–39% (Fig. 5a) and about 5% (Helmersson

et al. 2008), respectively. Assuming that embryogenic cul-

tures of Scots pine proliferate by a continuous cleavage-like

process, as has been suggested for other pine species

(Bozhkov et al. 1997), it is reasonable to assume that the

mechanism responsible for eliminating subordinate embryos

by PCD (Filonova et al. 2002) is retained in embryogenic

cultures, resulting in a much higher level of PCD in Scots pine

than in Norway spruce.

In cell line 3:10 the frequency of TUNEL-positive cells

decreased significantly during differentiation of early

somatic embryos (Fig. 5), while no significant decrease

was observed in cell line 12:12. An increase in PCD is

crucial during differentiation of early somatic embryos of

Norway spruce (Bozhkov et al. 2002; Helmersson et al.

2004). We assume that the lack of increased PCD during

differentiation of early somatic embryos in Scots pine is a

consequence of the high level of PCD during the prolif-

eration phase.

In order to assess if PCD is suppressed in Scots pine

somatic embryos carrying supernumerary suspensor cells,

we compared the frequency of TUNEL-positive cells in

embryos with normal morphology with that in embyos

carrying supernumerary suspensor cells in both cell line

3:10 and 12:12. We could not observe any clear differences

in the pattern of TUNEL-positive cells either between cell

lines or between normal embryos and embryos carrying

supernumerary suspensor cells (Fig. 6). This suggests that

the unbalanced ratio between the embryonal mass and the

suspensor is due to the formation of extra suspensor cells

rather than suppressed PCD.

Conclusion

The frequency of early somatic embryos with supernu-

merary suspensor cells is higher in Scots pine cell lines

producing cotyledonary embryos with an abnormal

morphology.

The proportion of Scots pine somatic embryos with

supernumerary suspensor cells increases after treatment

with the PAT inhibitor NPA, suggesting that the

Table 1 Effect of NPA treatment on the morphology of early somatic embryos

Cell line Treatment Total number of embryos Embryo morphology (%)

a b c d

12:12 Control 107 58 ± 2.7 2 ± 1.5 10 ± 4.6 31 ± 2.5

NPA 210** 28 ± 1.5** 9 ± 0.8** 21 ± 1.4* 42 ± 2.2*

3:10 Control 173 43 ± 3.8 6 ± 2.4 28 ± 0.9 24 ± 5.9

NPA 366** 29 ± 2.7** 6 ± 1.9 16 ± 3.5** 50 ± 2.3**

Embryogenic cultures of cell lines 12:12 and 3:10 were treated with 30 lM NPA during 2 weeks on pre-maturation medium and 2 weeks on

maturation medium. Presented data are based on scoring 200 aggregates in three independent experiments. The embryo morphology after

2 weeks on maturation was classified as type a, b, c or d as shown in Fig. 4. The frequency of embryos with various morphologies are based on

the total number of embryos ±SE. Within each cell line * denotes significant difference at the P \ 0.1 and ** denotes significant difference at

the P \ 0.05 between control and NPA treatment

Plant Cell Tiss Organ Cult (2012) 109:391–400 397

123

unbalanced ratio between the embryonal mass and the

suspensor is caused by disturbed PAT. The frequency of

TUNEL-positive cells is high in proliferating Scots pine

cultures. However, we could not observe any differences in

the pattern of TUNEL-positive cells between embryos with

normal morphology and embyos carrying supernumerary

suspensor cells, indicating that PCD is not suppressed in

embryos with supernumerary suspensor cells. We infer that

disturbed PAT stimulates division of the meristematic cells

adjacent to the tube cells and that this causes the formation

of supernumerary suspensor cells. The presence of

numerous suspensor cells probably affects the develop-

mental signaling within the embryo, which implies that the

origin of recalcitrance of Scots pine embryos in culture is

traceable to the events governing initial suspensor forma-

tion. Therefore, Scots pine and probably several other pine

species are out of phase with conifers where a limited size

of the suspensors is the normal phenotype.

Fig. 5 The frequency of

TUNEL-positive cells during

proliferation and differentiation

of early somatic embryos.

Samples were collected 2 weeks

after transfer to medium

containing PGRs (?PGR),

2 weeks after withdrawal of

PGRs (-PGR) and 2 weeks after

addition of ABA (ABA).

a Frequency of TUNEL-

positive cells in cell line 3:10

(black bars) and cell line 12:12

(grey bars) are based on the

total number of DAPI-stained

nuclei within each structure

(±SE). Presented data are based

on 15 randomly choosen

structures per treatment.

Treatments not connected by

same letter are significanly

different at the P B 0.05 level

according to t test. b Pattern of

PCD in cell line 3:10. ?PGR,

proliferating embryogenic cell

aggregate (stage 1a, Fig. 2a),

–PGR, slightly globular

structure with densely packed

cells (stage 1b, Fig. 2b), and

ABA, early embryos (stage 2,

Fig. 2d). Bars 100 lm

398 Plant Cell Tiss Organ Cult (2012) 109:391–400

123

Acknowledgments This work was supported by the Swedish

Research Council for Environment, Agricultural Sciences and Spatial

Planning. Silvia Valladares was supported by an Angeles Alvarino

postdoctoral fellowship from Xunta de Galicia (Spain).

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