ORIGINAL PAPER

Somatic embryogenesis in Alnus glutinosa (L.) Gaertn

Elena Corredoira • Silvia Valladares •

Ma Teresa Martınez • Ana Ma Vieitez •

Ma Carmen San Jose

Received: 11 March 2013 / Revised: 4 July 2013 / Accepted: 9 July 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract Induction of somatic embryos and plant

regeneration was demonstrated for the first time in Alnus

glutinosa. Somatic embryos were initiated from zygotic

embryos collected 1–3 weeks post-anthesis (WPA), i.e.,

when they were at globular or early cotyledonary stage and

were 0.5–1 mm in length. Induction frequency (16.6 %)

and the mean number of somatic embryos (4.5 embryos/

explant) were highest after culture of zygotic embryos,

collected at 3 WPA, on Murashige and Skoog medium

(MS) supplemented with 0.9-lM 2,4-dichlorophenoxy-

acetic acid and 2.22-lM benzyladenine (BA). No

embryogenic induction was observed on medium with BA

alone. Initial somatic embryos differentiated indirectly

from callus tissue formed at the surface of the zygotic

embryos. Embryogenic competence was maintained by

secondary embryogenesis, which was affected by explant

type, plant growth regulators and genotype. Secondary

embryogenesis was induced by culture of small groups of

whole somatic embryos or isolated cotyledon explants on

medium consisting of MS medium (half-strength ma-

cronutrients) supplemented with 0.44-lM BA. Histological

study of isolated cotyledon explants revealed that

secondary embryos developed directly from differentiated

embryogenic tissue on the surface of cotyledons. Somatic

embryos at successive stages of development, including

cotyledonary-stage embryos with shoot and root meris-

tems, were evident. For plantlet conversion, somatic

embryos were transferred to maturation medium supple-

mented with 3 % maltose, followed by 6 weeks of culture

in Woody Plant Medium supplemented with 0.44-lM BA

and 0.46-lM Zeatin (Z). This novel protocol appears

promising for mass propagation, conservation and genetic

transformation of black alder.

Keywords Black alder � Cones � Histology � Secondary

embryogenesis � Somatic embryos � Zygotic embryos

Introduction

Alders grow throughout the northern hemisphere in

woodland and riparian habitats. These species prefer cool

climates, although they can also withstand warmer tem-

peratures when water is adequate (Prada and Arizpe 2008).

The genus is thought to comprise approximately 30 spe-

cies, which are characterized by wind pollination, seed

dispersal by wind and water, rapid colonization of bare

ground and a relatively short life span. The male and

female flowers occur on separate catkins. The female

flowers are reddish-purple in color and develop into hard

cones containing seeds. Although alders are usually not the

focus of forest protection concerns in Europe, because they

are of minor importance to the forest economy, the eco-

logical value of the genus and its high value for land rec-

lamation and reforestation are well recognized (Cech

1998). Alders are capable of fixing atmospheric nitrogen

through symbiotic association with the actinomycete

Communicated by J. Carlson.

S. Valladares and E. Corredoira contributed equally to this work.

E. Corredoira (&) � S. Valladares � M. T. Martınez �A. M. Vieitez � M. C. San Jose

Department of Plant Physiology, Instituto de Investigaciones

Agrobiologicas de Galicia (CSIC), Apartado 122, 15780

Santiago de Compostela, Spain

e-mail: elenac@iiag.csic.es

123

Trees

DOI 10.1007/s00468-013-0907-8

Frankia alni (Oliveira et al. 2005), which forms nodules on

the tree roots. Alnus glutinosa (L.) Gaertn, also called

common alder, black alder or European black alder, is the

most common tree species in riparian forests. In addition to

the above-mentioned characteristics, black alder has a use

in flood control, stabilization of riverbanks and in func-

tioning of the river ecosystems (Claessens et al. 2010).

Black alder is also a host to a wide variety of moss and

lichen.

Unfortunately, alder populations are threatened by lethal

alder blight disease, which is caused by the pathogenic

fungus Phytophthora alni (Brasier et al. 2004).The disease

has been particularly destructive in the UK, but is also

found in Austria, Belgium, France, Germany, and Spain,

where it threatens natural riparian populations of alder

(Brasier et al. 1995; Cech 1998; Cavelier et al. 1999;

Streito et al. 2002; Jung and Blaschke 2004; Solla et al.

2010). Various attempts have been made to control the

disease through international research programmes, none

of which has yet succeeded in significantly halting its

spread or reducing its impact (Gibbs et al. 2003; Webber

et al. 2004). Genetic engineering may prove useful for

controlling alder blight disease. Regeneration of plants

from genetically transformed cells is a key step in devel-

oping a protocol for the genetic transformation of alder.

Somatic embryogenesis is an ideal regeneration system

for genetic transformation. This propagation technique is

defined as a process in which a bipolar structure, resem-

bling a zygotic embryo, develops from a somatic cell

without vascular connection to the original tissue (von

Arnold et al. 2002). Somatic embryogenesis, in which the

frequency of chimeras is low and the regeneration rate is

high, is more attractive than organogenesis as a plant

regeneration system (Gaj 2001). In addition, somatic

embryogenesis is considered the best in vitro propagation

method for most tree species (Merkle and Nairn 2005).

This technique could make a substantial contribution to the

conservation of alder species, not only as a means of pro-

ducing transgenic trees with resistance genes, but also for

mass propagation of the resistant genotypes produced by

traditional breeding programmes. However, as far as we

know, no studies have addressed the capacity of black alder

tissue to form somatic embryos. Alder species have only

been micropropagated by multiplication and rooting of

axillary shoots (Garton et al. 1981; Perinet and Lalonde

1983; Tremblay et al. 1986; Perinet and Tremblay 1987;

Barghchi 1988; Welander et al. 1989; Lall et al. 2005; San-

Jose et al. 2012, 2013).

The main objective of this study was, therefore, to

induce somatic embryogenesis from zygotic embryos (ZEs)

in Alnus glutinosa and to define a protocol for maintenance

of embryogenic lines and plantlet regeneration.

Materials and methods

Collection and sterilization of plant material

In the first experiment, cones were collected from three A.

glutinosa trees, of age 20–25 years, during August and

September 2011 (Table 1). The trees, which were desig-

nated Sarela 1, Sarela 2 and Sar 1, were growing in Garelas

park (Sarela 1 and Sarela 2) and Branas del Sar park (Sar 1)

in Santiago de Compostela (Spain). For the second exper-

iment, carried out in 2012, cones were collected from Sa-

rela 2 at weekly intervals during August and September,

i.e., approximately 1–6 weeks post-anthesis (WPA).

Anthesis is defined as the time at which approximately half

of the tree is in bloom.

The cones were washed in running tap water for 30 min

and surface sterilized by immersion for 1 min in 70 %

ethanol followed by immersion for 10 min in a 0.5 %

solution of free chlorine (Millipore� chlorine tablets) plus

1 % Tween�80. The cones were then rinsed twice with

sterile distilled water. After the sterilization process, the

seeds were removed from the cones, and intact zygotic

embryos were excised from seeds under a stereo micro-

scope in a flow laminar chamber. Isolated zygotic embryos

were then used as initial explants and were placed in

individual 20 9 150 mm culture tubes (one zygotic

embryo 9 tube) containing 16 ml of embryo induction

medium. A similar sterilization protocol has been used to

sterilize shoot tips and nodal segments of Paulownia

tomentosa (Corredoira et al. 2008).

Induction of somatic embryogenesis

In the first set of experiments, the explants were initially

cultured on induction medium consisting of Murashige and

Skoog medium (MS; Murashige and Skoog 1962) supple-

mented with 0.5 g/l casein hydrolyzate, 3 % sucrose, 0.7 %

agar (Difco Bacto, BD, USA), and 2.22-lM benzyladenine

(BA; Sigma-Aldrich, St. Louis, MO, USA) in combination

with 0.9, 2.26, 4.52 or 9.05 lM 2,4-dichlorophenoxyacetic

acid (2,4-D; Duchefa, The Netherlands). Before sterilizing

the medium, by autoclaving at 121 �C for 20 min, the pH

was adjusted to 5.7. For each tree, seeds were collected at

2–3 WPA, and 48 zygotic embryos were cultured for each

concentration of 2,4-D, so that there were 192 explants per

tree. In the second set of experiments, zygotic embryos

were collected from the Sarela 2 tree at 1–6 WPA and

cultured on MS plus 2.22-lM BA, with or without 2,4-D

(0.9 lM). Twenty-four explants were used for each com-

bination of induction medium and collection date, thus

providing 288 explants in total. In addition, on each col-

lection date, another set of 12 seeds and 12 zygotic

Trees

123

embryos was used to evaluate the mean length of seeds and

zygotic embryos.

After 4 weeks of culture on induction medium, the

explants were transferred to 300 ml jars (4 or 5 explants

per jar) containing 50 ml of expression medium consisting

of MS salts supplemented with 0.5 g/l casein hydrolyzate,

3 % sucrose, 0.7 % Difco bacto agar, and 0.44-lM BA.

The explants were transferred monthly to fresh medium

and were examined periodically to record the number of

explants exhibiting callus formation and somatic embryos.

Initial explants were maintained in darkness at 25 �C for

4 weeks and were then subjected to a 16-h photoperiod (30

lmol m-2 s-1 from cool-white fluorescent lamps) with

25 �C light and 20 �C dark temperatures. These conditions

were applied at all subsequent stages of the embryo pro-

liferation, maturation, and germination experiments.

Maintenance of embryogenic cultures

Isolated somatic embryos and embryogenic groups were

excised from the original explants and subcultured sepa-

rately on MS medium supplemented with 0.44-lM BA plus

0.54 lM a-naphthaleneacetic acid (NAA, Duchefa, The

Netherlands). On this medium, embryo proliferation by

secondary embryogenesis was low. Therefore, the capacity

for secondary embryogenesis was investigated by subcul-

turing embryo clumps of 1–3 whole somatic primary

embryos at globular to early cotyledonary stage (embryo-

genic line S1-1) on five proliferation media consisting of

MS medium (half-strength macronutrients) supplemented

with 3-mM glutamine, 3 % sucrose, 0.6 % agar (A-1296,

Sigma-Aldrich, St. Louis, MO, USA) and different com-

binations of plant growth regulators (PGRs) (Table 3).

Similar proliferation media have been used to maintain

different embryogenic lines of Quercus robur (Mallon

et al. 2012).

In a further experiment, the embryo proliferation

capacity of cotyledon explants isolated from somatic

embryos was evaluated using the same proliferation media,

except the medium supplemented with 0.54-lM NAA

(Table 4).

To investigate the influence of genotype, groups of 1–3

whole somatic embryos at globular-early cotyledonary

stage, derived from three different embryogenic lines

named S1-1 (derived from one seed of tree Sarela 1) and

S2-1 and S2-2 (derived from two seeds of tree Sarela 2)

were cultured on proliferation medium supplemented with

0.44-lM BA.

In each experiment, three replicate Petri dishes with six

explants per dish (18 explants in total) were used. Each

Petri dish was considered as a single replicate in a com-

pletely randomized block design, and the experiment was

repeated twice. In embryo proliferation experiments, the

number of explants forming secondary embryos, the

number of embryos per embryogenic explant, and the

multiplication coefficient (MC) were recorded after

6 weeks of culture. The multiplication coefficient was

defined as the product of the proportion of explants pro-

ducing secondary embryos and the mean number of

embryos per embryogenic explant. In the genotype

Table 1 Effect of 2,4-D concentration, tree genotype, and size of zygotic embryo on the induction of somatic embryogenesis in Alnus glutinosa

Tree Collection datea (WPA) Length of zygotic embryo (mm)b Surviving explants with callus (%)c Embryogenesis induction (%)

2,4-D (lM)

0.90 2.26 4.52 9.05

Sarela 1 2 0.8 ± 0.2 50.0 4.2 – – –

Sarela 2 2 0.6 ± 0.1 44.3 10.0 6.6 – –

Sar 1 2 0.9 ± 0.2 45.8 – – – –

Sar 1 3 1.3 ± 0.2 45.8 – – – –

Source of variation df v2 P

2,4-D concentration (A) 3 8.177 B0.05

Tree genotype (B) 2 7.873 B0.05

AXB 9 6.980 ns

For each tree and collection date, 192 explants were cultured

Influence of tree genotype and 2,4-D concentration on embryogenic induction was evaluated by the v2 test; ns not significant

–, no responsea Weeks post-anthesisb Values are means ± standard error of 12 zygotic embryosc After 4 weeks of culture

Trees

123

experiment, for each embryogenic line the total number of

somatic embryos per embryogenic explant and the number

of cotyledonary somatic embryos per embryogenic explant

were recorded after 6 weeks of culture.

Histological analysis

A histological study was performed during secondary

embryo proliferation. Samples were fixed in a mixture of

formalin, glacial acetic acid and 50 % ethanol [1:1:18 (v/v/

v)], dehydrated through a graded n-butanol series and

embedded in paraffin. Sections (8 lm) were cut on a Re-

ichert-Jung rotary microtome and were later stained with

periodic acid-Schiff (PAS)–naphthol blue-black, which is

commonly used to reveal total insoluble polysaccharides

and total protein content of the cells (O0Brien and McCully

1981). The stained sections were mounted with Euckit�,

and photomicrographs were taken with an Olympus DP71

digital camera fitted to a Nikon-FXA microscope.

Macroscopic features were observed in a stereo micro-

scope (Olympus SZX9) and photographed with an Olym-

pus DP10 digital camera.

Embryo maturation and germination

Somatic embryos, developed to the cotyledonary stage,

were carefully separated from the proliferation embryo-

genic explants and cultured on MS medium (half-strength

macronutrients) supplemented with either 6 % sucrose or

3 % maltose. Selection of these media was based on the

findings of previous studies on somatic embryo maturation

in other woody species (Iraqui and Tremblay 2001; Cor-

redoira et al. 2006a). After 5 weeks, somatic embryos were

transferred to glass jars (300 ml) with 25 ml of germination

medium consisting of Woody Plant Medium (WPM; Lloyd

and McCown 1981) supplemented with 2 % glucose, 0.6 %

agar, 0.44-lM BA and 0.46-lM Zeatin trans isomer (Z;

Duchefa, The Netherlands). This germination medium was

selected as the most efficient for the proliferation of axillary

shoot cultures in Alnus glutinosa (San Jose et al. 2013).

Twenty-five somatic embryos (five embryos per jar) were

used for each maturation treatment, and the experiment was

repeated twice. The numbers of somatic embryos that

developed roots only and those that successfully converted

into plantlets were recorded after 6 weeks of culture.

Statistical analysis

The influence of the 2,4-D concentration and tree origin on

the percentage of somatic embryogenesis (Table 1) was

analyzed using a v2 test (P \ 0.05). The interaction

between both factors was analyzed using a log-linear

model (P \ 0.05). The influence of the collection date on

the percentage of somatic embryogenesis was evaluated

using a v2 test (P \ 0.05) (Table 2). Factors influencing

secondary embryogenesis (PGRs and the embryogenic line

genotype) were analyzed by one-way analysis of variance

(ANOVA I). Test for normality and homogeneity of vari-

ance were performed prior to ANOVA, and the least sig-

nificant difference (LSD) or Dunnett’s T3 test (P B 0.05)

was used to compare means in the case of homogeneous or

non-homogeneous variances, respectively. All data were

analyzed using IBM SPSS statistical version 19.0 for

Windows (Chicago, USA).

Results

Induction of somatic embryogenesis

In the first set of experiments, zygotic embryos collected

during 2 WPA (Fig. 1a, b) from two (Sarela 1 and Sarela 2)

of the three trees tested, proved amenable to induction of

somatic embryogenesis via a straightforward procedure of

Table 2 Embryogenic response (percentage and mean number of somatic embryos) of Alnus glutinosa zygotic embryos (Sarela 2 tree) as a

function of collection date (weeks post-anthesis, WPA), length of seeds and of zygotic embryos, and developmental stage of the explants

Collection datea

(WPA)

Length of seed

(mm)bLength of zygotic

embryo (mm)bDevelopmental stage of the

zygotic embryo

Embryogenesis

induction (%)

Number somatic embryos

per explant

1 2.0 ± 0.1 0.5 ± 0.1 Globular 10.0 1.5 ± 0.3

2 2.3 ± 0.1 0.7 ± 0.1 Globular-early heart 11.1 2.0 ± 0.5

3 2.5 ± 0.2 1.0 ± 0.1 Early cotyledonary 16.6 4.5 ± 0.3

4 2.8 ± 0.1 1.6 ± 0.1 Cotyledonary – –

5 2.9 ± 0.1 2.2 ± 0.2 Cotyledonary – –

6 2.9 ± 0.1 2.5 ± 0.1 Cotyledonary – –

Influence of collection date on the embryogenic induction was evaluated by the v2 test (P B 0.01)

–, no responsea Weeks post-anthesisb Values are means ± standard error of 12 seeds or zygotic embryos

Trees

123

culturing these explants on medium containing the auxin

2,4-D and the cytokinin BA (Table 1).

The contamination frequency ranged between 5 and

50 % (Table 1) and was related to the tree of origin (e.g., in

Sar 1 contamination rate reached 50 %). After 4 weeks of

culture on induction medium, the proportion of surviving

explants with callus ranged from 44 to 50 % (Table 1).

After transfer of explants to the expression medium sup-

plemented with 0.44-lM BA and incubation under light

conditions, callus culture progressed, and somatic embryos

and embryogenic structures were generated at the surface

of white nodules that developed from the initial callus

(Fig. 1c, d). Somatic embryos appeared after 2–4 months

of culture on expression medium. Somatic embryo

Fig. 1 Somatic embryogenesis in zygotic embryos of Alnus

glutinosa. a, b Appearance of a seed (a) and of an immature zygotic

embryo (b) collected during 2 WPA; c somatic embryos initiated

from immature zygotic embryo cultured on induction medium

supplemented with 0.90 lM 2,4-D; d somatic embryo developed

from the upper surface of a cotyledon of the zygotic embryo;

e secondary somatic embryos after 6 weeks of the culture on the

proliferation medium; f somatic embryos developed from a isolated

cotyledon explant after 6 weeks on proliferation medium; g plantlet

from a somatic embryo maturated on maltose 3 % and germinated on

WPM medium with 0.44-lM BA and 0.46-lM Z; h acclimatized

somatic plantlets following 1 month in the growth chamber. Scale

bars a–e 1 mm and h 1 cm

Trees

123

development was asynchronous and somatic embryos at

different stages of development, from globular-through

cotyledonary-shaped embryos, appeared by this time.

Although somatic embryos were formed indirectly from

embryogenic calli, direct somatic embryogenesis also

occasionally occurred in parts of the zygotic embryos that

were not in contact with the medium. Somatic embryos

were morphologically similar to zygotic embryos. How-

ever, anomalous morphologies, including embryos with

fused cotyledons and embryos with more cotyledons than

the usual number, were also observed.

Induction of somatic embryos was significantly affected

by the 2,4-D concentration (P B 0.05) and tree genotype

(P B 0.05), but there was no interaction between the two

factors. Induction of somatic embryos was achieved in two

of the three trees evaluated, and the frequency of induction

was highest in the material collected from the Sarela 2 tree

(16.6 %). Somatic embryos were only initiated after cul-

ture on media supplemented with 0.90 and 2.26 lM 2,4-D

(Table 1), and the best results (10.0 %) were obtained with

0.90 lM 2,4-D.

A second set of experiments was carried out to identify

the ‘developmental window’ during which the induction of

somatic embryogenesis is possible in black alder. Based on

the results obtained in the first experiment, the best tree

(Sarela 2) and induction medium supplemented with

0.90 lM 2,4-D with or without 2.22-lM BA were chosen

for this induction experiment. Cones were collected from

Sarela 2 at weekly intervals between 1 and 6 WPA. On

each collection date, zygotic embryos were identified by

morphological measurements including size and develop-

mental stage (Table 2). For embryo induction in medium

including 0.9 lM 2,4-D, the collection date significantly

affected (P B 0.01) the induction response, although there

were no significant differences for collection dates of

1–3 WPA. Immature zygotic embryos collected 1–3 weeks

following anthesis proved to be the best explants for

induction of somatic embryogenesis (Table 2). During this

period, the zygotic embryos are at globular (0.5 mm) to

early cotyledonary stages (1 mm). The highest frequency

of embryogenic cultures was observed with zygotic

embryos at early cotyledonary stage and of mean length

1.0 mm. This type of explant also produced the highest

number of somatic embryos (4.5 per explants) (Table 2).

In cones collected in September (4–6 WPA), zygotic

embryos were at the cotyledonary stage and were charac-

terized by a marked and concomitant increase in the length

of the zygotic embryos (1.6–2.5 mm) and seeds

(2.8–2.9 mm). These embryos had lost their ability to

induce somatic embryos and most of them failed to pro-

duce callus and rapidly became necrotic (Table 2).

When initial explants (1–6 WPA) were cultured on

medium including BA alone, no SEs were produced.

However, the zygotic embryos collected during 6 WPA

(i.e., mature seeds) geminated successfully on this medium.

Maintenance of embryogenic cultures

Initial somatic embryos were easily separated from original

explants and were used to establish different embryogenic

lines of A. glutinosa, which were multiplied by secondary

embryogenesis (Fig. 1e). Each embryogenic line was

derived from a single zygotic embryo and, therefore, the

genotype of each line is different.

To optimize the proliferation rates, groups of 1–3 whole

somatic embryos (globular to early cotyledonary stage)

from embryogenic line S1-1 were cultured in media sup-

plemented with different combinations of PGRs and also

on PGR-free medium (Table 3). The PGRs treatment had a

significant effect on the frequency of explants that gener-

ated secondary embryos (P B 0.0001) and on the mean

number of secondary embryos (P B 0.0001) produced.

Although secondary embryos were formed on all prolifer-

ation media tested (44.4–100 %), the highest frequency

was obtained with medium supplemented with 0.44-lM

BA (100 %) (Table 3). Proliferation media supplemented

with NAA produced the smallest number of explants that

formed secondary embryos. The largest numbers of SEs

were obtained with 0.44-lM BA plus 0.46-lM Z (9.0 per

explant) and with 0.44-lM BA (8.7 per explant), although

the best medium in terms of somatic embryo productivity

(MC) was that containing only BA. When alder somatic

Table 3 Effect of different secondary proliferation media on the

percentage of explants producing secondary embryos, mean number

of embryos per explant and coefficient (MC) in whole somatic

embryos (embryogenic line S1-1) of Alnus glutinosa after 6 weeks of

culture on different proliferation media

PGRs (lM) Explants producing

secondary embryos

(%)a

Number of

somatic

embryos per

explantb

MCc

No PGR 91.7 ± 5.1b 5.3 ± 0.7b 4.8

BA 0.44 100.0 ± 0.0b 8.7 ± 0.6c 8.9

BA 0.44 ? Z 0.46 97.2 ± 2.5b 9.0 ± 0.5c 8.7

BA 0.44 ? NAA 0.54 83.3 ± 5.4b 5.7 ± 0.4b 4.7

NAA 0.54 44.4 ± 3.2a 3.1 ± 0.4a 1.4

F test P B 0.0001 P B 0.0001

Values are means ± standard error of three replicates from two

experiments, with six explants in each replicate

Within each column, values followed by the same letter are not sig-

nificantly different at P = 0.05, according to T3 Dunnett’s T3 testa or

LSD testb

c MC multiplication coefficient, defined as the proportion of explants

producing secondary embryos and the mean number of embryos per

embryogenic explant

Trees

123

embryos were subcultured on a PGR-free medium or on

medium supplemented with 0.44-lM BA plus 0.54-lM

NAA, the multiplication values were intermediate, while

proliferation medium with NAA alone yielded the lowest

values in terms of MC.

Morphological observations showed that secondary

somatic embryos mainly arose from the abaxial surface of

the cotyledonary region (Fig. 1f), and therefore in the

subsequent experiment, isolated cotyledons were tested for

embryo proliferation. Cotyledons were subcultured on the

same proliferation medium evaluated for whole somatic

embryos, with exception of the medium containing 0.54-

lM NAA (Table 4), which yielded the lowest proliferation

rates (Table 3). The frequency of isolated cotyledons pro-

ducing somatic embryos ranged from 31 to 88 %, with a

mean number of 2.9–6.0 embryos, and both parameters

were significantly affected by the treatment (Table 4).

Taking into account the three parameters evaluated, the

best results were achieved with the 0.44-lM BA treatment.

Once the 0.44-lM BA treatment was defined as the most

suitable medium for proliferation of somatic embryos of

embryogenic line S1-1, we evaluated its effectiveness for

the other embryogenic lines. The genotype and total

number of somatic embryos produced per explants were

not significantly different, although the genotype had a

significant effect on the number of cotyledonary somatic

embryos (P B 0.05) (Fig. 2).

Histological observations on secondary embryo forma-

tion revealed that embryos arise from a meristematic cell

zone differentiated from superficial layers of the cotyle-

dons. This meristematic zone is formed by cells with a

dense protein-rich protoplasm, high nucleoplasmic ratio

and that contain a few starch grains (Fig. 3a). Successive

divisions of these cells yielded direct formation of globu-

lar-stage embryos with visible protoderm (Fig. 3b) fol-

lowed by development of oblong (Fig. 3c), torpedo

(Fig. 3d), and cotyledonary stages (Fig. 3e, f). The origin

of secondary embryos was direct without any apparent

callus phase, and embryos at all stages of development

were observed on the same embryogenic explant. Longi-

tudinal sections of cotyledonary stage embryos showed a

well-defined embryonic axis, with both shoot and root

meristems, and a procambium system without any con-

nections to the vascular system of the primary somatic

embryo (Fig. 3f).

Embryo maturation and germination

Experiments were carried out to investigate the plant

regeneration ability of black alder somatic embryos. The

germination ability was affected by the carbohydrate

source of the maturation medium (3 % maltose or 6 %

sucrose). The best results were obtained with somatic

embryos maturated on 3 % maltose, which gave rise to

20 % of embryos exhibiting only root development and a

plantlet conversion rate of 8.0 %. The embryos developed

true shoots with new leaves and roots (Fig. 2g). Somatic

embryos that did not develop into plantlets became

abnormal and formed calli or secondary embryos on their

surfaces before eventual disorganization. Plantlets were

successfully acclimatized and the survival rate was 100 %

(Fig. 1h).

Table 4 Effect of different secondary proliferation media on the

percentage of explants producing secondary embryos, mean number

of embryos per explant and coefficient (MC) in isolated cotyledon

explants (embryogenic line S1-1) of Alnus glutinosa after 6 weeks of

culture on different proliferation media

PGRs (lM) Isolated cotyledon

producing secondary

embryos (%)

Number of

somatic embryos

per cotyledon

MCa

No PGR 31.7 ± 6.7a 2.9 ± 0.3a 0.92

BA 0.44 88.3 ± 2.4b 6.0 ± 0.6c 5.33

BA 0.44 ? Z 0.46 76.1 ± 9.3b 5.2 ± 0.6cb 3.96

BA 0.44 ? NAA

0.54

47.8 ± 11.6a 3.8 ± 0.9ab 1.82

F test P B 0.01 P B 0.05

Values are means ± standard error of three replicates from two

experiments with six cotyledons in each replicate

Within each column, values followed by the same letter are not sig-

nificantly different at P = 0.05 level according to LSD testa MC multiplication coefficient defined as the product of the pro-

portion of explants producing secondary embryos and the mean

number of embryos per embryogenic explant

Fig. 2 Effect of genotype on secondary embryo production (total

number of somatic embryos and number of cotyledonary somatic

embryos) of three embryogenic lines of Alnus glutinosa. Results of

statistical analysis of the data in the figure are presented in the text.

Scale bars represent standard errors

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Discussion

Induction of somatic embryos has been obtained for the

first time in a species of the genus Alnus using immature

zygotic embryos as the starting material. The main factors

determining the induction of somatic embryogenesis in

black alder are collection date (closely related to the

developmental stage of zygotic embryos), concentration of

PGRs and genotype. Alder somatic embryos were obtained

by culture of zygotic embryos on medium supplemented

with 2,4-D. The important role of 2,4-D in the induction of

somatic embryogenesis when zygotic embryo was used as

Fig. 3 Histological observation of secondary somatic embryogenesis

of Alnus glutinosa. a Transverse section of a cotyledon explant after

15 days of culture on proliferation medium showing numerous

divisions of epidermal cells (arrow heads) and typical embryogenic

cells with dense cytoplasm, a higher nucleoplasmic ratio and starch

grains (arrows). b Cotyledonary region showing histodifferentiation

of globular-stage embryo from the meristematic (m) surface layers;

c cotyledonary region with differentiation of oblong-stage embryo.

Note mitotic division (arrow); d cotyledonary region with differen-

tiation of torpedo-stage embryo (t). e Cotyledonary embryos arising

from the meristematic surface layers of primary cotyledon. Note the

globular somatic embryo (gl) developed from cotyledonary somatic

embryo; f cotyledonary somatic embryo showing shoot (sm) and root

meristems (rm), and vascular system (vs)

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initial explants has been reported for many woody species

(Carraway and Merkle 1997; Canhoto et al. 1999; Capuana

et al. 2007; Vengadesan and Pijut 2009; Rocha et al. 2012).

This auxinic herbicide appears to create a double effect by

acting directly as an auxin while also triggering the pro-

duction of endogenous indole-3-acetic acid and even by

acting as a stress factor (Gaj 2004). Parallel activation of

auxin and stress signaling may be key events in cellular

adaptation via reprogramming the gene expression pattern,

cellular metabolism, and physiology (Pasternak et al.

2002). Thomas and Jimenez (2006) suggested that early

stages of somatic embryogenesis are characterized by the

induction of many stress-related genes, leading to the

hypothesis that SE is an extreme stress response in cultured

plant cells (Dudits et al. 1995).

In the present study, the highest frequency of somatic

embryogenesis was obtained in induction medium supple-

mented with 0.90 lM 2,4-D. This concentration of auxin

has also been found to be the most effective for inducing

somatic embryogenesis in zygotic embryos of elm (Cor-

redoira et al. 2002). Feher (2006) pointed out that auxins

and cytokinins are often used to induce somatic embryos

because they regulate the cell cycle and trigger cell divi-

sion. In alder, somatic embryogenesis took place in med-

ium containing 2,4-D and BA, but not in medium

supplemented with BA alone, suggesting that the presence

of 2,4-D was critical for embryogenesis. Similarly, Cor-

redoira et al. (2002) found that culture of zygotic embryos

of Ulmus minor and U. glabra on medium supplemented

with BA was not effective for induction of somatic

embryos. Although in some species, cytokinins rather than

auxins were found to be effective for induction of somatic

embryos (Kim et al. 2003; Chung et al. 2005; Pacheco et al.

2007), somatic embryogenesis is usually induced in the

presence of auxins alone or in combination with cytokinins.

The type of explant and the development stage appear to

be important factors determining the embryogenic capacity

(Gaj 2004). In black alder, there is a short period during

development of the zygotic embryo when it is possible to

establish embryogenic cultures. Zygotic embryos collected

in August (1–3 WPA), i.e., when they were at the globular

to early cotyledonary stages and of length 0.5–1 mm, were

responsive. The induction frequencies and the mean num-

ber of somatic embryos were highest when the zygotic

embryos were collected at early cotyledonary stage, before

deposition of storage reserves. We have previously found

that for elm, the best embryogenic induction rates were

achieved with zygotic embryos at cotyledonary stage,

before storage proteins began to accumulate (Corredoira

et al. 2002). In other species, such as Cercis canadensis

(Trigiano et al. 1999) and Tilia cordata (Karkonen 2000),

the zygotic embryos are embryogenically competent only

prior to maturation, and the appearance of significant

protein reserves signals a decline in embryogenic compe-

tence. The length of alder zygotic embryos collected in

September (4–6 WPA) increased considerably due to

elongation of cotyledons and the accumulation of reserve

substances (associated with seed maturation), and these

embryos were not able to initiate somatic embryos. Once

histodifferentiation of alder zygotic embryos up to the

cotyledonary stage has occurred (approximately 3 WPA),

the subsequent maturation during the remaining weeks

involves rapid elongation of the zygotic embryo, and

therefore length may be a good parameter for selecting

explants for initiating somatic embryogenesis.

Somatic embryos of black alder showed the potential to

initiate new cycles of somatic embryogenesis by repetitive

or secondary embryogenesis. Although proliferation med-

ium lacking plant growth regulators has been used to

maintain the embryogenic ability of various woody species

(Hernandez et al. 2003; Mauri and Manzanera 2005; Pintos

et al. 2008), in alder the application of embryo proliferation

medium supplemented with cytokinin (BA with or without

Z) significantly increased embryo production. Cytokinins

have been shown to promote secondary embryogenesis in

several woody species (Jimenez 2005). BA is commonly

used alone or in combination with other growth regulators,

mainly auxins, for the proliferation of somatic embryos

(Corredoira et al. 2003a, 2012; Valladares et al. 2006;

Cerezo et al. 2011); however, in black alder, the results

obtained with NAA alone or in combination with BA

indicated that exogenously supplied auxin was not neces-

sary for secondary embryo proliferation. Similarly, Mallon

et al. (2012) reported that the addition of NAA to the

proliferation medium has a negative effect on the multi-

plication of pedunculate oak somatic embryos. There are

evidences that once embryogenesis is induced, the role of

auxin changes and embryos begin to synthesize their own

auxins and, thus, require less additional auxin (Zimmerman

1993).

Secondary embryos were also obtained from isolated

cotyledons cultured on different proliferation media.

However, the multiplication coefficient values were lower

than those obtained when whole somatic embryos were

used as explants. The differences in proliferation ability

suggest that isolated cotyledons cannot synthesize the

appropriate PGRs or they do not produce sufficient levels

of PGRs. In addition, the number of embryogenic cells

involved in generating new secondary embryos is possibly

higher in whole somatic embryos than in isolated cotyledon

explants.

As expected, genotype had a clear influence on the

induction of black alder somatic embryos and also on the

embryo proliferation stage as the three different embryo-

genic lines tested yielded significantly different numbers of

cotyledonary somatic embryos. Hernandez et al. (2003)

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also observed a genotype effect on the secondary embryo

multiplication of Quercus suber somatic embryos, based on

significant differences in the relative increase in fresh

weight and number of ‘detachable’ cotyledonary embryos.

We have also found significant differences in the prolif-

eration rates of four embryogenic lines derived from elm

zygotic embryos (Corredoira et al. 2003b).

Determination of the origin of the cell or groups of cells

involved in plant regeneration events is essential for the use

of somatic embryogenesis in clonal propagation and in

genetic transformation protocols (Rocha et al. 2012). In

black alder, histological observations showed that sec-

ondary embryos differentiated from the meristematic zones

originated from surface layers of cotyledon explants. These

meristematic zones were composed by cells with similar

embryogenic attributes as those reported for other woody

species, such as coffee (Quiroz-Figueroa et al. 2002),

pedunculate oak (Corredoira et al. 2006b), peach palm

(Steinmacher et al. 2007), and white oak (Corredoira et al.

2012). Black alder secondary embryos originated directly

from cotyledonary tissues, whereas the somatic embryos

originated from initial zygotic embryo explants have an

indirect pattern of differentiation and arise from callus

tissue formed from these zygotic embryos. Direct regen-

eration of secondary embryos is important for both plant

propagation and genetic transformation studies because

direct regeneration is less likely to generate somaclonal

variation than indirect regeneration from callus tissue (Gaj

2001).

In general, maturation of somatic embryos can be

achieved through treatment with abscisic acid (ABA) and/

or osmotic agents (such as high concentrations of sugars or

sugar alcohols), which induce water stress in the culture

medium (von Arnold et al. 2002). In black alder, somatic

plantlets were obtained after culture of somatic embryos on

maturation medium supplemented with 3 % maltose,

although conversion rates were low. Therefore, further

research on the maturation and germination of alder

somatic embryos is still required. In several woody species,

embryo conversion and germination are the limiting steps

in embryogenic systems (Merkle et al. 2005; Cerezo et al.

2011; Vieitez et al. 2012).

The protocol defined in this study may help to enhance

the propagation of Alnus glutinosa and other alder species,

and it will also provide a regeneration system for future use

in genetic manipulation for the introduction of genes con-

ferring resistance to pathogens such as Phytophthora alni.

However, application of the black alder embryogenic sys-

tem as a standard procedure or for use in genetic engi-

neering is still limited by the low plantlet conversion rate.

Efforts towards improving embryo maturation and

increasing the conversion rate should be made in future

research.

Acknowledgments We thank Ma Jose Cernadas and Jose Carlos

Suarez for their excellent technical and laboratory assistance. This

research was partially funded by INLUDES (Diputacion Provincial de

Lugo) and Xunta de Galicia (Spain) through Project

09MRU002400PR.

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