ORIGINAL PAPER

Improved organogenic capacity of shoot cultures from maturepedunculate oak trees through somatic embryogenesisas rejuvenation technique

Teresa Martınez • Nieves Vidal • Antonio Ballester •

Ana M. Vieitez

Received: 1 April 2011 / Revised: 30 June 2011 / Accepted: 8 July 2011 / Published online: 24 July 2011

� Springer-Verlag 2011

Abstract Theoretically, complete rejuvenation of mature

trees should occur through somatic embryogenesis, how-

ever, this has not been extensively studied. The main

objective of the present study was to increase the efficiency

of in vitro clonal propagation for mature Quercus robur

(100–300 years old), by induction of somatic embryogene-

sis as rejuvenation step prior to establishment of shoot cul-

ture through micropropagation of somatic embryo-derived

plantlets. Shoot culture lines of ‘‘mature’’ origin were

established from epicormic shoots of two centenarian oak

genotypes (Sainza and CR-0) and maintained by axillary

shoot proliferation. Embryogenic lines were also initiated

from epicormic leaf explants of the same genotypes and

maintained by secondary somatic embryogenesis. Although

the frequency of somatic embryo conversion into plantlets

was low in pedunculate oak, shoot culture lines could be

established and maintained by axillary branching from

several germinated somatic embryos. For each genotype and

shoot culture line of the two origins (mature tree and somatic

plantlets), shoot multiplication rate and elongation as well as

rooting ability parameters were compared. Compared with

‘‘mature-origin’’ shoot cultures and after more than one year

propagation in vitro, shoot lines established from somatic

plantlets produced a significantly higher proportion of

elongated, rootable shoots (from 26.0–31.6 to 36.8–40.5%)

with increased rooting ability (from 3.3–45.6% to

23.2–89.8%). In the case of 300-year-old Sainza genotype

such a high organogenic capacity was similar to shoot

cultures initiated from basal sprouts. Basal sprouts are

considered as ‘‘mature’’ material that retains juvenile char-

acteristics compared with epicormic shoots forced from

crown branches. Somatic embryogenesis only slightly

improved plant regeneration of shoot cultures from basal

sprouts, thus validating their use as ‘‘juvenile control’’. The

present results provide evidence that some rejuvenation

occurred during the process of somatic embryogenesis and

resulted in improved shoot growth and rooting of somatic

embryo-derived culture compared with ‘‘mature’’ shoot

culture. The results reported in this study might be useful in

embryogenic systems with low plant conversion rates. The

proposed experimental model might also be useful in finding

molecular markers of plant ontogeny.

Keywords Micropropagation � Physiological aging �Quercus robur � Rooting � Somatic plantlets

Introduction

Multivarietal forestry, defined as the clonal deployment of

tested tree varieties in plantation forestry, may dramatically

increase forest productivity over any conventionally used

strategies (Klimaszewska et al. 2007; Weng et al. 2010).

An effective clonal propagation method must be available

for multivarietal forestry, but mature trees exhibiting

genetically desirable traits are generally recalcitrant to

vegetative propagation, as a consequence of ontogenetic

maturation. Micropropagation by both organogenesis and

somatic embryogenesis (SE) is influenced by phase change

and the culture environment; the former is a poorly

understood phenomenon, particularly the reversal of phase

change from mature to juvenile stages, which occurs in the

Communicated by K. Klimaszewska.

T. Martınez � N. Vidal � A. Ballester � A. M. Vieitez (&)

Instituto de Investigaciones Agrobiologicas de Galicia, CSIC,

Avda. de Vigo, s/n, Apartado 122, 15780 Santiago de

Compostela, Spain

e-mail: amvieitez@iiag.csic.es

123

Trees (2012) 26:321–330

DOI 10.1007/s00468-011-0594-2

sexual process (von Aderkas and Bonga 2000). The pos-

sible causes of recalcitrance in clonal propagation of trees

as well as some potential solutions have recently been

addressed (Bonga et al. 2010). The induction of SE in vitro

is in principle the most efficient method for inducing

rejuvenation (Pierik 1990) and may be the only method of

regenerating truly juvenile propagules (Bonga et al. 2010).

However, rejuvenation via organogenesis was reported in

Sequoiadendron giganteum (Monteuuis and Bon 1989). If

this is true, plants derived from somatic embryos should

exhibit juvenile characteristics, including a high capacity

for micropropagation through organogenesis (Carron et al.

1995). The complete rejuvenation of plants obtained from

somatic embryos induced in explants from adult trees has

still to be proven (Hernandez et al. 2011). Indeed, data

demonstrating rejuvenation in plants derived from germi-

nated somatic embryos of mature tree origin are scarce. SE

has been induced in mature specimens of Hevea brasili-

ensis and rejuvenation was reflected in the re-acquisition of

the micropropagation capacity (Carron et al. 1995; Perrin

et al. 1997). The ability of somatic plants and SE-derived

clones to undergo organogenesis in vitro has become of

great interest in Theobroma cacao (Traore et al. 2003).

Efforts are being made to establish in vitro regeneration

systems for clonal propagation of pedunculate oak (Quer-

cus robur L.). Micropropagation of adult oak trees based

on axillary branching is feasible if tissues retaining phys-

iological juvenile characteristics, such as stump sprouts or

epicormic shoots collected at the base of the trunk, are used

as the source of initial explants (Chalupa 2000; Vidal et al.

2003). When such material is not available, a procedure

based on forced flushing of branch segments can be used to

obtain epicormic shoots, which may be sufficiently rein-

vigorated for use as a source of reactive explants (Vieitez

et al. 1994; Ballester et al. 2009). This procedure allows the

establishment and proliferation of shoot cultures derived

from mature trees, although the rooting ability of these

cultures is not satisfactory for all oak genotypes.

Theoretically, SE is a more efficient procedure for clo-

nal mass propagation than micropropagation by axillary

shoot induction and proliferation in forest trees. The

induction of SE in mature trees has been reported for only a

limited number of conifers (Klimaszewska et al. 2011) and

broad-leaved species, including pedunculate oak. In the

latter species, SE has been established from leaf explants

(Toribio et al. 2004; Valladares et al. 2006) and shoot tip

explants (San-Jose et al. 2010) derived from centenarian

trees. The fact that oak embryogenic lines can be main-

tained by repetitive embryogenesis and that a number of

somatic embryos become germinated indicates that this

process may become a very powerful approach for clonal

propagation of this valuable genus, which has been found

to be recalcitrant in vegetative propagation (Merkle and

Nairn 2005). However, major difficulties must still be

overcome in order to make this method commercially

viable in oak, particularly when cultures originate from

mature trees. Once induced, the main problem reported for

oak SE is a very low rate of conversion of somatic embryos

into plants, which remains difficult in most genotypes,

resulting in limited production of plants (Vieitez et al.

2011). However, SE could be used as a rejuvenation step in

vegetative propagation of mature trees and the somatic

plants could be multiplied through rooting of axillary

shoots. So far, this approach has not been yet investigated

in oak.

Vidal et al. (2003) established shoot cultures initiated

from basal sprouts (BS line) and crown branches (C line) of

a mature Q. robur tree (Sainza). These authors defined the

shoot line originated from basal sprouts as mature material

retaining juvenile characteristics compared with C line,

based on morphological features and rooting ability.

Therefore the Sainza-BS line could serve as a suitable

reference material for evaluating the degree of juvenility

attained by shoot lines originated from somatic embryo-

derived plantlets of the same genotype.

The main objective of the present study was to test the

hypothesis of true rejuvenation via the induction of SE. A

rejuvenation procedure was designed whereby the induc-

tion of SE from mature oak played a central role, and was

integrated into micropropagation system by using the

somatic embryo-derived plants. The decline in adventitious

rooting capacity in forest tree cuttings is generally one of

the most dramatic effects of maturation and it is the most

widely used morphological and physiological marker to

track maturation (Greenwood et al. 2001). In this study, a

specific objective was to compare the morphogenetic

capacity (axillary shoot proliferation and growth, rooting

rates, root growth and number) of pedunculate oak clones

established in vitro from selected mature trees and from

plants derived from somatic embryos induced from the

same trees. The present study demonstrates that efficient

propagation of mature trees can be achieved by rejuvena-

tion through SE, even when very poor conversion of

somatic embryos into plantlets is the limiting factor in

recovering clonal plants.

Material and methods

Initiation of shoot cultures from mature trees

In vitro axillary shoot proliferation cultures were estab-

lished in 2008 from two centenarian oak trees, denoted

Sainza (around 300 years) and CR-0 (around 100 years),

growing in Galicia (northwestern Spain). Shoot cultures

were initiated from explants from different topophysical

322 Trees (2012) 26:321–330

123

positions including basal sprouts (Sainza-BS line) and

epicormic shoots obtained by forced flushing of branch

segments collected from the crown (Sainza-C and CR-0

lines). Basal sprouts were only available for Sainza. As

basal sprouts retain juvenile physiological characteristics

compared with epicormic shoots from crown branches

(Vidal et al. 2003), we refer to these isogenic shoot cultures

as ‘‘control juvenile’’ and ‘‘mature’’, respectively. In the

case of shoot culture induction from epicormic shoots

(Vieitez et al. 1994; Ballester et al. 2009), nodal segments

and shoot tips were used as initial explants, which gave rise

to bud flushing and growing shoots after transfer of the

explants to fresh medium every 2 weeks during the

6–10 weeks culture period on the initial shoot development

medium. The medium was GD (Gresshoff and Doy 1972)

supplemented with 2.22 lM benzylaminopurine (BA),

6.5 g/l Vitroagar (Pronadisa, Spain) and 30 g/l sucrose. All

of the shoot culture lines had been cultured since 2008 in

shoot proliferation medium consisting of the initial shoot

development medium but with BA reduced to 0.44 lM.

During the subcultures, decapitated shoot explants (2 cm

long), i.e. with the apical 2 mm shoot apex removed, were

placed horizontally in 500 ml glass jars (6 shoots per jar)

containing 70 ml of proliferation medium. After 2 weeks

explants were transferred onto fresh medium, with BA

reduced to 0.22 lM also for 2 weeks resulting in a 4-week

multiplication cycle. At the end of this period, newly

developed shoots were excised for the next subculture

cycle (Vieitez et al. 1994; Ballester et al. 2009). Shoot

multiplication rate and growth (shoot length) were evalu-

ated for each genotype and shoot line.

The pH of all media was adjusted to 5.6–5.7 before

autoclaving at 115�C for 20 min. All cultures were incu-

bated under a 16-h photoperiod provided by cool-white

fluorescent lamps at the photon flux density of

50–60 lmol m-2 s-1, at 25�C light/20�C dark.

Initiation of shoot cultures from somatic plantlets

regenerated from mature tree-derived embryogenic

lines

Embryogenic lines used in this study were initiated

between 2004 and 2005, from leaf explants of basal sprouts

or forced epicormic shoots developed on crown branch

segments. Material was collected from the same mature

oak trees (Sainza and CR-0) used to establish ‘‘mature’’

shoot culture (Toribio et al. 2004; Valladares et al. 2006).

Within the Sainza genotype, two embryogenic lines were

thus differentiated as those induced from leaf explants of

basal sprouts (Sainza-BS-SE) or epicormic shoots forced

from crown branches (Sainza-C-SE and CR-0-SE). In the

case of Sainza-C-SE material, two shoot culture lines were

established from two different germinated embryos, and

were designated Sainza-C-SE-1 and Sainza-C-SE-2.

Another centenarian genotype (B-17) was similarly used to

produce a third embryogenic line initiated from epicormic

shoots (B-17-SE). These embryogenic lines had been

maintained by secondary embryogenesis with sequential

subculture on embryo proliferation medium at 6-week

intervals since 2004 (B-17-SE) and 2005 (Sainza-BS-SE,

Sainza-C-SE and CR-0-SE) (Valladares et al. 2006).

Embryo maturation and germination were performed fol-

lowing procedures previously reported for Q. robur SE

(Martınez et al. 2008). Briefly, prior to germination,

somatic embryos were cultured for 4 weeks on maturation

medium consisting of MS (Murashige and Skoog 1962)

medium with half strength macronutrients and supple-

mented with 6% (w/v) sorbitol. Following maturation,

somatic embryos (6–8 mm in length) of the four embryo-

genic lines were transferred to germination medium that

differed from the maturation medium in containing

0.44 lM BA and no sorbitol. After 8 weeks of culture in

the germination medium, plantlet conversion was obtained

when embryos exhibited both root and shoot development.

In 2008, plantlets derived from germinating somatic

embryos, were used to establish shoot culture lines

(Fig. 1a, b). The shoot developed in the epicotyl region (at

least 1.5 cm long) was cut off and used as explant (with the

apical 2 mm removed) for multiplication by axillary bud

development, on shoot proliferation medium. Shoot cul-

tures from somatic embryo-derived plants were thus

obtained and multiplied following the same procedure used

for shoot lines derived from mature trees, with each line

derived from one somatic plant.

Determination of shoot proliferation rate, growth

and rooting ability

Shoot cultures derived from mature trees (‘‘control juve-

nile’’ BS and ‘‘mature’’ C lines) or somatic plantlets

(BS-SE and C-SE lines) were first evaluated for shoot

multiplication rate and growth after at least one year of

shoot culture initiation and successive monthly subcultures

that yielded sufficient number of stabilized culture.

Although the culture line derived from a B-17 somatic

plantlet was included in this study, the shoot culture line

from the mature tree was not established in vitro from the

donor tree because of a problem related to its identification

in the field. The proliferation rate and rooting ability of line

B-17-C-SE were therefore not compared with the coun-

terpart ‘‘mature’’ line.

For each genotype and shoot culture line, shoot multi-

plication rates were determined at the end of the 4-week

multiplication cycle by recording the following variables:

the number of shoots longer than 4 mm per explant (total

shoot number); the number of shoots longer than 14 mm

Trees (2012) 26:321–330 323

123

(rootable shoots) per explant and the length of the tallest

shoot produced by each explant. From the rootable shoots

and total shoot number we deduced the frequency of roo-

table shoots produced by any shoot culture line.

Shoot culture lines of different origin (‘‘control juvenile’’

from basal sprouts, ‘‘mature’’ from epicormic shoots or

somatic plantlets) were in a second step used as a source of

rootable shoots ([14 mm) to estimate the rooting rate.

Shoots of 1.5–2.0 cm in length were isolated from cultures

proliferated for 12–18 months and transferred onto root

induction medium. The medium was GD with macronutri-

ents reduced to one third strength, 30 g/l sucrose, 6.5 g/l

Vitroagar and 122.5 lM indole-3-butyric acid (IBA). After

24 h (B-17 line) or after 48 h (Sainza and CR-0 lines), shoots

were transferred to the same medium deprived of auxin and

supplemented with 0.4% (w/v) activated charcoal (Vieitez

et al. 2009). In these experiments, six shoots were cultured in

100 ml glass jars each containing 30 ml of rooting medium.

The rooting response was determined 1 month after the

onset of auxin treatment, by calculating the rooting

Fig. 1 Plant regeneration from

shoot culture lines derived from

somatic plantlets. Somatic

plantlets obtained from

germinated somatic embryos of

lines B-17 (a) and Sainza BS

(b) after 8 weeks of culture in

germination medium. The

shoots of these plantlets were

excised and used to establish

shoot culture lines B-17-C-SE

and Sainza-BS-SE, respectively.

c Shoot culture lines derived

from a mature tree, CR-0-C

(left), and from a somatic

plantlet, CR-0-C-SE (right),after 4 weeks of culture on

shoot proliferation medium.

CR-0 lines have been

maintained after 17 monthly

subcultures since initiation.

d Rooting response of shoots

isolated from the shoot culture

line Sainza-BS-SE, 1 month

after the beginning of auxin

treatment. Sainza-BS-SE (e) and

Sainza-C-SE-1 (f) acclimatized

plants after 4-month growth

in the greenhouse. Scale barsa–d 1 cm; e, f 2 cm

324 Trees (2012) 26:321–330

123

percentage and counting the number of roots per rooted

shoot and the length of the longest root produced by each

rooted shoot.

Sampling and data analysis

For each combination genotype 9 line origin (BS, BS-SE,

C, C-SE), shoot multiplication rate and growth were esti-

mated. Six replicate 500 ml jars (6 explants per jar) were

used per shoot line, and the experiment was repeated twice.

Thirty explants per treatment were used in the case of

rooting (6 explants per 100 ml jar) and the experiment was

repeated three times (Table 2). For evaluation the kinetics

of rooting (Fig. 2), two replicates with 18 shoots per rep-

licate (6 shoots per jar) were used for each rooting date. For

each genotype the data were subjected to analysis of var-

iance between treatments followed by pair wise mean

comparison with the Tukey’s HSD (Honestly Significant

difference) test at the P B 0.05 level, when more than two

treatments were available (Sainza genotype). Data on

plants grown in the greenhouse represent mean ± standard

error.

Results

Shoot proliferation, growth and rooting

Shoot cultures were initiated from basal sprouts (Sainza

genotype) and forced epicormic shoots from crown bran-

ches (Sainza and CR-0 genotypes). After 3–4 transfers of

primary explants on initial shoot development medium,

shoots of at least 2 cm in length were excised from primary

explants and proliferated following subculture cycles of

4 weeks. Only newly developed shoots exhibiting vigorous

growth were used in successive subcultures. Following this

procedure the ‘‘juvenile control’’ and ‘‘mature’’ shoot

cultures became stabilized (achieving uniform, continuous

shoot growth) after 4 (CR-0-C and Sainza-BS lines) or

5 months (Sainza-C line).

As expected, Sainza shoot cultures derived from the

crown (C line) appeared more mature than the ‘‘juvenile

control’’ obtained from basal sprouts (BS line), which

ontogenetically is younger. Significantly lower frequency

of rootable shoots (26 vs. 42%) and lower length of the

longest shoot (19.4 vs. 23.9 mm) were observed, whereas

shoot number was significantly higher in Sainza-C com-

pared with Sainza-BS line (Table 1).

Compared to Sainza-BS derived from basal sprouts,

Sainza-BS-SE line produced similar number of shoots and

rootable shoots per explant. The length of the longest shoot

is increased but the difference was not significant

(Table 1). The number of shoots produced by Sainza-C-SE

lines was not significantly different from Sainza-C line. In

contrast, significantly higher number and frequencies of

elongated shoots ([14 mm), and longer shoots were

observed in the lines from somatic plantlets obtained from

crown branches (Sainza-C-SE-1 and Sainza-C-SE-2), sug-

gesting that some rejuvenation occurred. Similar values for

all the parameters were obtained in both Sainza-C-SE lines,

and the frequency of rootable shoots ([14 mm) and their

length (the longest shoot) were not significantly different

from the Sainza-BS line although the mean number of

rootable shoots per explant was significantly higher in

Sainza-C-SE-1 line compared with Sainza-BS line. This

suggests that the variables related to shoot elongation were

enhanced in cultures derived from somatic embryo-derived

plants of mature origin, and were similar to those of the

physiologically juvenile line, Sainza-BS (Table 1).

Differences in rooting capacity were observed in the

different Sainza lines (Table 2). The rooting ability of the

‘‘juvenile control’’ Sainza-BS shoots was significantly

higher (71.5%) than that of Sainza-C line (only 3.3%),

confirming the high difference in rooting capacity previ-

ously reported for these materials (Vidal et al. 2003).

Considering shoot lines derived from somatic plantlets, the

rooting rate was significantly higher (92.7%) in the Sainza-

BS-SE line (Fig. 1d) than in the Sainza-BS line, which has

physiologically juvenile characteristics and a relatively

high rooting ability. However, the improved rooting fre-

quency of the Sainza-BS-SE line seemed to indicate that

some rejuvenation occurred also after using SE in this

‘‘juvenile control’’ material (Sainza-BS). Thus, both BS

and BS-SE lines were finally considered as ‘‘juvenile

controls’’. Lines Sainza-C-SE-1 and Sainza-C-SE-2

derived from two different somatic plantlets rooted simi-

larly (no significant differences) and better than the mature

Sainza-C line; however, these two Sainza-C-SE lines

exhibited quite low rooting frequencies compared with

Sainza-BS. The highest mean root numbers were produced

* **

* * *

0

20

40

60

80

100

5 6 7 8 9 10 11 12 13 14 15 16 17 18

Months

Ro

oti

ng

(%

)

BS-SE

C-SE-1

Fig. 2 Kinetics of rooting ability of Sainza-BS-SE and Sainza-C-SE-

1 lines after 5–18 monthly subcultures. The bar represents the

standard error of means of two replicates with 18 shoots per replicate

(6 shoots/jar). Within the same rooting date, asterisk denotes

significant differences at P \ 0.05

Trees (2012) 26:321–330 325

123

in Sainza-BS lines, whereas no significant differences were

observed between Sainza-C lines (from mature tree and

from somatic plantlets). Root length was not significantly

affected by shoot line origin.

Although the rooting experiments (Table 2) were car-

ried out after stock shoot lines had been propagated in vitro

for more than one year by monthly subcultures, we also

evaluated the kinetics of rooting of the Sainza-C-SE-1 and

Sainza-BS-SE lines during 5–18 months of successive

subcultures (Fig. 2). In line C-SE-1, rooting rate was ini-

tially increasing to reach 74% after 7 months of subculture

(no significantly different from Sainza-BS-SE), and later

stabilized around 23%. Despite the decrease in rooting

ability, the rooting frequencies of Sainza-C-SE-1 and also

Sainza-C-SE-2 were significantly greater than that of Sai-

nza-C of mature tree origin, which may indicate the

acquisition of at least a certain level of rejuvenation for the

shoots derived from somatic plantlets. In contrast, kinetics

of rooting in Sainza-BS-SE line was consistently high

(Fig. 2) suggesting retention of juvenility in this line.

In CR-0 genotype, similar number of shoots was

obtained in lines derived from somatic plantlets (CR-0-C-

Table 1 Shoot number and length estimated in micropropagated cultures of three centenarian Q. robur trees (Sainza, CR-0 and B-17) derived

from ‘‘control juvenile’’ basal sprouts (BS), epicormic shoots from crown branches (C) and/or somatic embryo-derived plants (SE)

Genotype Origin Number of

shoots/explant

Number of shoots

[14 (mm)/explant

Percentage shoots

[14 (mm)

Longest shoot

length (mm)/explant

Sainza BS 5.5 ± 0.5 c 2.3 ± 0.1 bc 42.0 ± 2.5 a 23.9 ± 0.9 a

Sainza BS-SE 5.9 ± 0.3 bc 2.4 ± 0.2 abc 42.5 ± 2.4 a 26.4 ± 1.1 a

Sainza C 7.4 ± 0.5 ab 2.0 ± 0.2 c 26.0 ± 1.2 b 19.4 ± 0.4 b

Sainza C-SE-1 8.7 ± 0.5 a 3.2 ± 0.2 a 37.6 ± 2.9 a 22.8 ± 1.0 ab

Sainza C-SE-2 8.3 ± 0.3 a 3.0 ± 0.3 ab 36.8 ± 3.3 a 25.1 ± 0.8 a

F-test P \ 0.001 P \ 0.001 P \ 0.001 P \ 0.001

CR-0 C 7.5 ± 0.5 2.4 ± 0.2 31.6 ± 2.3 20.9 ± 0.6

CR-0 C-SE 7.5 ± 0.2 3.0 ± 0.2 40.5 ± 1.6 28.3 ± 1.3

F-test NS P \ 0.05 P \ 0.01 P \ 0.001

B-17 C-SE 5.2 ± 0.3 2.6 ± 0.1 49.4 ± 2.1 24.6 ± 0.9

Within each column and genotype, values followed by different letters differ significantly (P \ 0.05) according to Tukey’s HSD test

Data represent mean ± standard error of two replicate experiments with 36 shoots per replicate (6 shoots/jar). Experiments 1 and 2 have been

performed after 14 and 17 monthly subcultures since initiation, respectively

Table 2 Rooting ability of shoots in micropropagated cultures of three centenarian Q. robur trees (Sainza, CR-0 and B-17) derived from

‘‘control juvenile’’ basal sprouts (BS), epicormic shoots from crown branches (C) and/or somatic embryo-derived plants (SE)

Genotype Origin Rooting % Root number

per rooted shoot

Longest root length

per rooted shoot (mm)

Sainza BS 71.5 ± 1.8 b 3.0 ± 0.3 ab 21.9 ± 0.8

Sainza BS-SE 92.7 ± 5.0 a 4.3 ± 0.5 a 24.5 ± 5.1

Sainza C 3.3 ± 1.9 d 1.0 ± 0.0 c 15.5 ± 7.5

Sainza C-SE-1 23.2 ± 1.9 c 1.4 ± 0.4 bc 19.9 ± 5.4

Sainza C-SE-2 24.3 ± 2.9 c 1.3 ± 0.1 bc 22.0 ± 3.5

F-test P \ 0.001 P \ 0.001 NS

CR-0 C 45.6 ± 9.5 1.7 ± 0.1 23.4 ± 1.7

CR-0 C-SE 89.8 ± 3.5 2.5 ± 0.3 35.1 ± 3.2

F-test P \ 0.05 NS P \ 0.05

B-17 C-SE 90.0 ± 2.4 2.1 ± 0.2 16.9 ± 0.5

Within each column and genotype, values followed by different letters differ significantly (P \ 0.05), according to Tukey’s HSD test

Data represent mean ± standard error of three replicates with 30 shoots per replicate (6 shoots/jar). The three experiments have been performed

after 13, 15 and 18 monthly subcultures since initiation

326 Trees (2012) 26:321–330

123

SE) and the mature tree (CR-0-C), although the former

produced significantly higher number and frequency of

rootable shoots and the longest shoots (Table 1; Fig. 1c).

Multiplication rate (total shoot number per explant) was

not a suitable marker for maturity in this genotype; how-

ever, similarly to Sainza genotype, shoot length was sig-

nificantly enhanced in CR-0-C-SE cultures and was also

associated with development of shoots with longer inter-

nodes (Fig. 1c), which may reflect a higher juvenility

degree for the CR-0-C-SE line, in comparison with that of

crown epicormic shoot from mature trees.

Rooting percentage of CR-0-C-SE line was significantly

higher than in CR-0-C line from the mature material

(Table 2). Although the mean root number and root length

were also higher in the line of somatic embryo origin,

significant differences were only observed in the root

length, which may be associated with earlier emergence of

root primordia. Furthermore, the high rooting frequency of

CR-0-C-SE shoots was maintained at 87% when the con-

centration of IBA was reduced to 15 mg/l in the root

induction medium, whereas the rooting frequency of the

CR-0-C line decreased to 27% after the 15 mg/l IBA

treatment. This suggests that differences in rooting ability

of the two lines may be related to physiological differences

in the control of auxin activity. These data reinforce the

hypothesis of rejuvenation.

The B-17-C-SE line produced quite long shoots

(Table 1) and had long internodes (data not shown). In this

line the percentage of rootable shoots and the longest shoot

length were closer to the values obtained in Sainza-BS

‘‘juvenile control’’ and CR-0-SE lines than those of mature

material (C lines). Furthermore, the high rooting percentage

obtained in B-17-C-SE is similar to rooting rates achieved

by Sainza-BS-SE (‘‘juvenile control’’) and CR-0-SE lines

suggesting an improvement in rooting capacity due to SE

involvement in the establishment of this line. The fact that

there were not significant differences (P = 0.931) among

these lines of different genotypes for rooting ability

strengthens the rejuvenation likelihood of B-17-C-SE line.

However, because of technical reasons we could not

establish shoot culture line from the B-17 mature tree and

hence we could not demonstrate that these parameters were

associated with the acquisition of juvenility.

Acclimatization and growth of somatic plants

Rooted plantlets of Sainza-BS-SE and Sainza-C-SE-1 were

transplanted to pots containing sterilized peat:perlite (3:1)

for acclimatization in a phytotron for 6–8 weeks and then

grown in the greenhouse. After 4 months in the greenhouse,

significant differences (P \ 0.001) in plant growth between

Sainza-BS-SE and Sainza-C-SE-1 origins were observed,

with mean shoot length of 21.7 ± 1.3 cm in the former and

10.9 ± 0.8 cm in the latter (Fig. 1e, f). These differences

were more prominent after 12 months in the greenhouse:

40.4 ± 2.2 cm for BS-SE plants and 23.0 ± 2.4 cm for

C-SE-1 plants (P \ 0.001). Significant differences

(P \ 0.001) were also found in the mean length of inter-

nodes reaching 1.45 ± 0.07 cm and 0.97 ± 0.06 cm for

plants of BS and C origins, respectively.

Discussion

Recalcitrance in clonal propagation is still a major problem

for many tree species at the adult phase including mature

oaks. To our knowledge, this is the first study demon-

strating that shoot proliferation and rooting ability of shoot

culture derived from mature oak trees can be stimulated

through the likely rejuvenation achieved by means of SE.

The establishment and maintenance of shoot cultures

derived from crown branches of mature CR-0 and Sainza

trees confirmed the suitability of combining forced flushing

of branch segments and culture of decapitated shoots in a

stressful horizontal position to achieve stabilized prolifer-

ating shoot cultures from adult Quercus sp. (Vieitez et al.

1994, 2009).

Data obtained on shoot proliferation and rooting of

Sainza-BS line and Sainza-C line confirm results already

published by Vidal et al. (2003) who reported differences

in the degree of juvenility between BS line originated from

basal sprouts and C line derived from the crown of the

Sainza mature tree. These authors determined that shoot

and internodal sections were larger in BS than in C shoots

and the former had greater rooting capacity (73.4%) than

the latter (2.2%). Despite the time elapsed between Sainza

shoot lines used by Vidal et al. (2003) and those established

in 2008 and used in the present study, the multiplication

and shoot growth rates, and rooting ability were consistent

in Sainza cultures, which indicates a high degree of sta-

bility over time in these proliferating cultures derived from

two different topophysical positions.

Shoot lines from somatic embryo-derived plantlets of

Sainza and CR-0 trees (Sainza-C-SE-1 and -C-SE-2; CR-0-

SE) exhibited improved shoot and root regeneration com-

pared with C shoot cultures (Sainza-C and CR-0-C).

Rejuvenation was reflected in a greater elongation of the

shoots produced and a greater proportion of rootable

shoots. In addition, C-SE lines showed similar shoot pro-

duction and length compared with the ‘‘juvenile control’’

(Sainza BS). McGowran et al. (1998) reported that shoot

length and vigor of Q. robur shoot cultures of juvenile

origin tended to be higher than those of shoot cultures of

mature origin. The period of rapid shoot growth was

associated with the juvenile phase whereas reduced rate of

growth occurred at maturation (Greenwood 1987;

Trees (2012) 26:321–330 327

123

McGowran et al. 1998). In this study, a significant

improvement in rooting rates was obtained in stock shoot

cultures of somatic plantlet origin, in comparison with the

mature tree counterparts. Interestingly, in the case of basal

sprouts, SE-derived shoot culture (Sainza-BS-SE) did not

show any significant difference of measured parameters

except for higher rooting percentage compared with Sai-

nza-BS culture. This could indicate that SE only slightly

improved rooting capabilities of shoot culture from basal

sprouts, thus validating its use as juvenile control. Rooting

frequencies of 43–55% were obtained in shoots from two

H. brasiliensis genotypes regenerated from somatic

embryo-derived plants, whereas it was impossible to ini-

tiate rooting in shoots obtained from the mature plant

material (Carron et al. 1995). Rooting rates of oak shoot

lines from somatic plantlets were greater (around 90%)

than those achieved in Hevea genotypes (after five monthly

subcultures), with the exception of Sainza-C-SE lines,

which exhibited decreasing rooting ability as a function of

time (Fig. 2). Similar rooting compared with ‘‘juvenile

control’’ (BS) is expected if complete rejuvenation occur-

red. However, the two Sainza-C-SE lines rooted signifi-

cantly better than the mature Sainza C line. It appears that

the physiological rejuvenation theoretically associated with

SE also occurred in Sainza materials, although this was

especially evident during the first subcultures of the Sai-

nza- C-SE-1 stock lines. Aging is a cause of decreased

organogenic capabilities including from culture initiated

from juvenile material such as embryogenic lines (Breton

et al. 2006). On the basis of shoot production and growth

and the rooting rates recorded, at least a certain degree of

physiological rejuvenation/reinvigoration may have been

induced in the Sainza-C-SE plant materials, although the

relationship between genotype and rooting capacity should

also be highlighted. The results obtained within Sainza

lines seem to indicate that there may be a gradient of

physiological juvenility, with Sainza-BS-SE displaying the

maximum degree of juvenility and Sainza-C the lowest.

Mankessi et al. (2009) suggested that salient time-rela-

ted fluctuations in adventitious rooting capacity in two

Eucalyptus clones of mature origin may be influenced by

genotype-related endogenous rhythms. However, the Sai-

nza material (Sainza-BS and Sainza-C) showed a strong in

vitro stability, with very similar shoot growth and rooting

responses in shoot culture lines used by Vidal et al. (2003),

and those established in 2008 used in the present study.

Currently, all Sainza lines established in 2008, maintain

similar shoot multiplication and growth, and rooting rates

(results not shown) than those indicated in Tables 1 and 2.

Given that the Sainza tree is approximately 300 years old,

this may influence the rapid loss of rooting competence of

the somatic-embryo-derived lines (Sainza-C-SE-1 and -C-

SE-2) as consequence of a possible aging effect occurring

during the successive subcultures. The reduced shoot

growth and internode length observed in greenhouse grown

plants, derived from mature C-SE-1 line compared with

plants of BS-SE origin (juvenile control), reinforces the

likely negative aging effect in plants of mature origin.

Apart from a possible genotypic effect, ontogenetic aging

may have had more pronounced effect in lines derived

from somatic plantlets from the old Sainza than in lines

derived from somatic plantlets of CR-0 or B-17.

The differences in shoot development of somatic plants

(derived from Sainza-BS-SE and Sainza-C-SE-1 lines),

grown in the greenhouse may be a consequence of the dif-

ferences in physiological juvenility attained by plants of

these two origins. As well, an influence of the different mean

root number produced by these plantlets in vitro (Table 2)

could also cause this difference, especially during the first

period of growth after transplanting. In contrast, no differ-

ences were found between cork oak somatic plants of adult

(epicormic shoots) and juvenile (seedlings from halfsib

acorns of the adult trees) origin after the first growing season

in the field (Celestino et al. 2007). However, apart from a

possible genotypic effect, a different experimental model

was used in cork oak in which plants were derived from

germinated somatic embryos, whereas in this study somatic

embryo-derived clones and rooted plants were used.

The actual degree of physiological rejuvenation dis-

played by the lines derived from somatic embryo requires

further study. An estimate of the level of rejuvenation

achieved after certain treatment should not be based only

on the in vitro micropropagation criteria but also on studies

of biochemical (Perrin et al. 1997), and genetic and/or

epigenetic markers of juvenility. In a study on biochemical

markers to differentiate mature (Sainza-C) and juvenile

(Sainza-BS) shoot lines, Vidal et al. (2003) detected higher

amounts of endogenous indoleacetic acid (IAA) in C shoots

than in BS shoots during the rooting process. The authors

concluded that endogenous IAA concentration does not

seem to be the limiting factor in rooting of mature oak

material, and it is possible that a lack of appropriate

receptors prevents IAA from acting on the cells of this

material. In addition, a full-length cDNA clone (QrCPE)

was differentially expressed in Sainza-BS and Sainza-C

shoot lines (Gil et al. 2003). Accumulation of QrCPE

mRNA was higher in C shoots than in BS shoots.

Expression of this gene was also investigated during the in

vitro rooting of Sainza shoots (Covelo et al. 2009). These

authors showed a greater expression of QrCPE in Sainza-

BS shoots treated with auxin than in auxin-treated Sainza-

C shoots during the first days of adventitious root induc-

tion. Changes in DNA methylation were associated with

maturational changes during ontogeny of S. giganteum

(Monteuuis et al. 2008). Measurements of global DNA

methylation or polyamin content in Pinus nigra was

328 Trees (2012) 26:321–330

123

correlated with embryogenic potential (Noceda et al.

2009). Qualitative differences in juvenile and mature

Acacia mangium, as demonstrated by methylation-sensitive

amplification polymorphism (MSAP) techniques, have also

been reported (Baurens et al. 2004). Similar results have

been mentioned for the biological characterization

of young and aged embryogenic cultures of P. pinaster

(Klimaszewska et al. 2009). Identification of genes that are

expressed at high levels in rejuvenated tissues will

undoubtedly aid in the development of tissue-culture con-

ditions that may eventually overcome the problem of

recalcitrance (Bonga et al. 2010). The experimental model

proposed in the present study could help in the identifica-

tion of molecular markers of plant ontogeny.

The results reported here showed that when there is low

conversion rates of somatic embryos to plants, the few that

converted could be micropropagated by axillary shoot

proliferation. The establishment of somatic embryo-

derived shoot lines with greater number of rootable shoots

and higher rooting ability than mature origin shoot culture

may improve the efficiency of oak micropropagation.

Acknowledgments We thank M. J. Cernadas and C. Garcıa for

technical assistance. This research was partially funded by Xunta de

Galicia and Consejo Superior de Investigaciones Cientıficas (Spain)

through the projects 09MRU002400PR and PIE200940I011,

respectively.

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