Factors influencing axillary shoot proliferation and ... - Tree Physiology

Tree Physiology 25, 477–486 

© 2005 Heron Publishing—Victoria, Canada 

Factors influencing axillary shoot proliferation and adventitious 

budding in cedar 

BEGOÑA RENAU-MORATA, 1 JAVIER OLLERO, 1 ISABEL ARRILLAGA 1 and JUAN 

SEGURA 1,2 

1 

Departamento de Biología Vegetal, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andrés Estelléss/n, 46100-Burjasot (Valencia), 

Spain 

2 

Corresponding author (juan.segura@uv.es) 

Received July 29, 2004; accepted October 2, 2004; published online February 1, 2005 

Summary We developed procedures for in vitro cloning of 

Cedrus atlantica Manetti and C. libani A. Rich explants from 

juvenile and mature plants. Explant size was one determinant 

of the frequency of axillary bud break in both species. Shoot 

tips and nodal explants mainly developed calli, whereas bud 

sprouting occurred in defoliated microcuttings cultured on a 

modified Murashige and Skoog medium without growth regulators. 

Isolation and continuous subculture of sprouted buds on 

the same medium allowed cloning of microcuttings from C. atlantica 

and C. libani seedlings and bicentennial C. libani trees, 

thus providing a desirable alternative for multiplying mature 

trees that have demonstrated superior characteristics. We also 

report adventitious bud differentiation from isolated embryos 

of C. atlantica. Neither auxin treatments nor other methods 

tested, including infection with Agrobacterium rhizogenes, 

were effective in inducing root initiation. 

Keywords: Cedrus atlantica, Cedrus libani, isolated embryos, 

microcuttings, tissue culture. 

Introduction 

Deforestation due to human activity, accompanied by depletion 

of forest tree genetic resources, is occurring in many parts 

of the world. Thus, there is an urgent need to conserve forest 

ecosystems for both environmental and economic reasons (Fenning 

and Gershenzon 2002). To maintain and sustain forest 

trees, conventional breeding and silvicultural approaches have 

been exploited for propagation and improvement, but treebreeding 

efforts are restricted to the most valuable and fastest-growing 

species. Such methods are limited because trees 

are generally slow-growing, long-lived, sexually self-incompatible 

and highly heterozygous. Plant tissue culture offers an 

alternative approach for rapidly propagating and improving 

forest stock, as well as a means to overcome the impediments 

associated with conventional tree breeding (Altman 2003, Giri 

et al. 2004). However, other features inherent to the biology of 

forest trees—such as the recalcitrance of tissues from mature 

trees to in vitro manipulation—continue to challenge researchers 

developing tissue culture protocols for commercial 

tree production (Merkle and Dean 2000). 

Cedar forests are of great importance in the Mediterranean 

area. In Morocco, Cedrus atlantica Manetti is the main source 

of timber, fuel wood and other valuable products (Benchekroun 

1994). In Turkey and Lebanon, timber from Cedrus 

libani A. Rich has been used for centuries in construction, 

sculpture and crafts (Khuri et al. 2000, Talhouk et al. 2001). In 

addition, both species are cultivated worldwide as ornamental 

trees (Toth 1980, Hapla et al. 2000, Brunetti et al. 2001). Cedars 

have been found difficult to propagate asexually and have 

been propagated by grafting only under experimental conditions 

(Siniscalco 1994). However, tissue culture techniques 

may give rise to methods that allow efficient propagation of 

cedar species. A few reports on in vitro propagation of cedars 

are available (Bhatnagar et al. 1983, Abourouh and Najim 

1990, Piola and Rohr 1996, Piola et al. 1998, 1999, Khuri et al. 

2000) but none of them provide clear protocols yielding reproducible 

results that would provide a basis for commercial production. 

Our objective was to develop methods for the in vitro 

propagation of C. libani and C. atlantica from juvenile and 

adult trees. 

Materials and methods 

Juvenile explants 

Shoot apices (0.2–0.4 cm), nodal segments (with 2–3 axillary 

buds) and defoliated shoots (1–2 cm in length, hereafter referred 

to as microcuttings) from C. libani and C. atlantica 

seedlings were used as initial explants. Seedlings were raised 

from seeds (from Chiltern Seeds, Ulverston, England, or Intersemillas, 

Quart de Poblet, Spain) that had been soaked 

(1–3 min) in 1% NaClO and stratified for 1 month in darkness 

at 4 °C in plastic trays containing a sterile mixture of peat moss 

and perlite (1:1, v/v), moistened with half-strength Hoagland 

and Arnon (1950) nutrient solution with 0.1 g l –1 8-hydroxyquinolin 

as fungicide. Subsequently, trays were transferred to 

a growth chamber at 25 ± 1 °C, with a 16-h photoperiod of 

70 µmol m –2 s –1 and 70% relative humidity. After 2 to 

3 months, stems (1–3 cm in length) were excised from the 

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478 RENAU-MORATA, OLLERO, ARRILLAGA AND SEGURA 

seedlings and surface sterilized in 3% NaClO with 0.1% 

Tween-20 for 15 min or 0.1% HgCl2 for 3 min, followed by 

four 5-min washes in sterile distilled water. Needles were removed 

with a razor blade at 1–2 mm above the needle–node 

junction, and these initial explants were cultured on different 

nutrient media. 

Freshly isolated C. atlantica embryos were used as primary 

explants in some experiments. Seeds were imbibed for 4 h in 

sterile distilled water, surface-sterilized for 30 min in 70% ethanol 

and rinsed in sterile distilled water; the mature embryos 

were then dissected out of the seedcoat and female gametophyte. 

Only undamaged embryos that were firm and white to 

pale yellow in color were cultured. 

Mature explants 

Actively growing shoots (3–5 cm long) were collected from 

several trees growing outdoors, including trees in a 20–30year-old 

forest plantation of C. libani and C. atlantica located 

in Arbúcies, Girona, Spain; 15–20-year-old C. libani trees in a 

natural forest located in El Shouf, Lebanon; and 200-year-old 

C. libani trees growing at the Reading University campus, 

Reading, U.K. All material was collected in the spring (May to 

early June). After the leaves were removed, stems were immersed 

in 1% NaClO for 20 min, rinsed for 8 h in running tap 

water, submerged for 15 min in an aqueous solution of 1% 

benomyl, and kept at 4–6 °C for 1–10 days. Shoots were then 

surface sterilized with 0.1% HgCl2 as described for the juvenile 

explants. Initial explants, meristematic domes plus one or 

two pairs of leaf primordia (0.6–1.0 mm), apical shoots (6– 

7 mm) and microcuttings were isolated under sterile conditions 

and cultured on different nutrient media. Unless otherwise 

stated, initial explants were chosen at random from different 

donor plants. 

Media and culture conditions 

The basal media tested were: Murashige and Skoog (1962) 

medium, with macronutrients reduced to a half (MS/2) or a 

quarter (MS/4) of their original concentration; modified SH 

medium (MSH), with Schenk and Hildebrandt (1972) macronutrients 

and MS micronutrients and vitamins; modified BF 

medium (MBF), with Boulay and Franclet (1977) salts and 

MS vitamins; MSBN/2 medium, with half-strength MS salts 

and half-strength Bourgin and Nitsch (1967) vitamins and 

Skoog (1944) amino acids; MSBN/4 (half-strength MSBN/2 

medium); and WPM medium (Lloyd and McCown 1980). All 

media contained 3% sucrose except for MSBN/2 which contained 

2% sucrose. Culture media were supplemented with 

growth regulators (indoleacetic acid (IAA), naphthaleneacetic 

acid (NAA), benzyladenine (BA), zeatin (Z) and thidiazuron 

(TDZ)) as specified. In some experiments, conifer-derived activated 

charcoal (Sigma, St. Louis, MO), calcium gluconate, 

triacontanol, paclobutrazol, pectimorf (a mix of oligopectins 

from citric cell walls, kindly provided by Dr. Juan Carlos 

Cabrera, INCA, Cuba), coumarin or phosphoric acid were 

used. Several agar brands were tested: Difco-Bacto (0.7%; 

Sparks, MD), Sigma phytagel (0.2%), Sigma A-1296 (1%) 

and Pronadisa (0.8%; Torrejón de Ardoz, Madrid, Spain). The 

TREE PHYSIOLOGY VOLUME 25, 2005 

pH was adjusted to 5.8 before autoclaving for 20 min at 120 °C 

(10 5 Pa). Except for TDZ and Z which were sterilized by filtration, 

growth regulators and ancillary compounds were added 

to the medium before autoclaving. 

Culture vessels included 15 × 100-mm petri dishes and 150 

× 25-mm glass tubes covered with polypropylene closures 

(Bellco, Vineland, NJ), each containing 25 ml of agar-solidified 

nutrient medium; Sigma jars with 100 ml of medium and 

glass jars with 50 ml of medium, both covered with sun-cap 

closures (Sigma); and 100-ml pots. In some experiments, 

explants were cultured, with continuous agitation (110 rpm), 

in 125-ml Erlenmeyer flasks (containing 25 ml of liquid medium) 

capped with Bellco stainless steel closures. 

Unless otherwise specified, cultures were kept in growth 

chambers at 26 ± 2 °C and a 16-h photoperiod provided by fluorescent 

lamps (70 µmol m –2 s –1 irradiance at culture level). 

Axillary shoot proliferation from juvenile explants 

Nutrient media, explant type, growth regulators and agar 

brands Shoot apices and three types of nodal explants (upper 

(containing buds of the first verticil, immediately below the 

apex); central (second verticil); and basal (third verticil, immediately 

above the cotyledonary node)) were initially dipped for 

3 or 12 h in a sterile aqueous solution of 100 µM TDZ or BA, 

and then transferred individually to glass tubes containing 

agar-solidified MSH, MBF or MSBN/2 nutrient medium with 

or without BA (2.2 or 9 µM), 0.45 µM TDZ, 10 –14 µM IAA or 

combinations of the three types of growth regulators. Control 

explants were maintained for 3 or 12 h in sterile distilled water. 

The following brands of agar were tested: Difco-Bacto, Phytagel, 

Pronadisa and A-1296. Explant-derived organogenic calli 

were isolated, transferred to their respective media without 

growth regulators and kept at 26 or 30 °C. At least 13 apical or 

nodal explants were cultured for each treatment. Total culture 

time was 40 days. 

Explant size, incubation temperature and cytokinin type In 

a first experiment, shoot apices and microcuttings were cultured 

on medium with and without 9 µM BA and maintained at 

30 °C. In a second series of experiments, both entire and decapitated 

microcuttings were cultured on medium without growth 

regulators or supplemented with 2.2 µM BA or 2.2 µM Z or 

0.45 µM TDZ. Cultures were kept at 26 or 30 °C. In all experiments, 

microcuttings were placed vertically in glass tubes containing 

MSBN/2 medium solidified with A-1296 agar. In addition, 

microcuttings from C. libani proliferating cultures were 

isolated and cultured on the same medium with or without 

2.2 µM Z and kept at 26 °C. Between 10 and 14 replications 

were prepared for each treatment. Cultures were examined for 

survival and sprouting percentages and mean number of axillary 

shoots per sprouted microcutting. Culture time was 40– 

60 days. 

Axillary shoot proliferation from mature explants 

In a first experiment, meristematic domes of C. atlantica and 

C. libani trees from Arbúcies, Spain, were cultured for 30 days 

on Difco-Bacto agar-solidified MSH or MS/2 medium supple- 



mented with BA (0, 2.2, 4.4 or 8.8 µM), alone or in combination 

with NAA (0, 0.5, or 5.4 µM). Subsequently, explants 

were transferred to their respective media without growth regulators 

for another 30 days. Twenty explants were cultured per 

treatment. 

In a second experiment, shoot apices from C. libani trees 

growing in Arbúcies were cultured for 45 days in glass tubes 

containing A-1296 agar-solidified MSBN/2 medium supplemented 

with BA (0, 0.44, 2.22 and 4.44 µM), NAA (0 and 

0.05 µM), or combinations of both types of growth regulators. 

Subsequently, explants were transferred to their respective 

media without growth regulators for another 45 days. Twentysix 

explants were cultured per treatment. 

In a third experiment, microcuttings from C. libani trees 

growing in El Shouf, Lebanon, were cultured in glass tubes 

containing A-1296 agar-solidified MSBN/2 medium without 

growth regulators. To obtain enough material for further studies, 

developing axillary shoots were routinely isolated from 

the elongated microcuttings and subcultured on the same medium. 

Shoots isolated from these proliferating cultures were 

first cultured for 60 days in glass tubes containing WPM medium 

or MSBN/2 medium supplemented with 0 or 0.45 µM 

TDZ or 2.22 µM Z. Subsequently, explants were transferred to 

their respective media without growth regulators. At least 10 

microcuttings were cultured per treatment. 

To test the effect of genotype, 50 to 140 microcuttings from 

six 200-year-old C. libani trees (genotypes AL1 to AL6), 

growing in Reading, U.K., were cultured on A-1296 agar-solidified 

MSBN/2 medium without growth regulators. Axillary 

shoots that formed on these explants were subcultured on the 

same medium to promote their elongation and the proliferation 

of new buds. This procedure was repeated about every 

2 months for 2 years. 

In all experiments, cultures were examined for survival and 

sprouting percentages and the mean number of axillary shoots 

per sprouted explant. 

Adventitious bud differentiation from explants of C. atlantica 

In a first experiment, excised needles from 3-month-old C. libani 

and C. atlantica seedlings were cultured (adaxial surface 

to the medium) in petri dishes containing MSH medium with 

or without 0.5 µM BA. Each treatment contained 20 replicates 

(20 dishes with five explants each) and culture time was 

60 days. 

In a second experiment, freshly isolated C. atlantica embryos 

were cultured in petri dishes containing MSBN/2 medium 

supplemented with BA or Z (0, 2.2, 4.4, 6.6 or 9.0 µM). 

After 60 days, embryos were transferred to the same medium 

with or without 4.4 µM Z. Proliferating buds were isolated and 

transferred to fresh medium without growth regulators. Embryos 

were also cultured for 60 days on MSBN/2 or WPM medium 

supplemented with Z (4.4, 9.0 or 18.0 µM), and then 

subcultured as described previously. Each treatment contained 

five replicates (five dishes with five embryos each). 

In all experiments, media were solidified with A-1296 agar 

and cultures were examined for percent of explants with callus 

or buds and number of adventitious buds per explant. 

SHOOT PROLIFERATION IN CEDAR CULTURES 479 

TREE PHYSIOLOGY ONLINE at http://heronpublishing.com 

Rooting 

Both axillary and adventitious shoots (longer than 1 cm) isolated 

from C. atlantica and C. libani proliferating cultures, 

previously maintained for 1 month on MSBN/2 without 

growth regulators, were used for rooting experiments. Unless 

otherwise stated, shoots with trimmed basal needles were cultured 

in glass tubes containing MBN/2 medium solidified with 

A-1296 agar and cultures were maintained in a 16-h photoperiod. 

In a first series of experiments, shoots were either cultured 

for 7, 15 or 30 days on medium containing IBA or IAA (0, 0.3, 

2.5, 5, 10, 25 or 50 µM), or dipped in IBA (2.5 or 5 mM) in the 

presence or absence of 1 mM NAA for 1 s and 5 min, respectively. 

Alternatively, the basal ends of the shoots were treated 

with rooting powders (2.5 mM IBA in talcum or RAIFORT SF 

from RIBA Quimiagra SL, Granollers (Barcelona), Spain, a 

commercial formulation containing 53.7 mM NAA and fungicides). 

Sterile support systems (filter paper bridges on liquid 

medium, a mixture (1:1) of peat moss and perlite or vermiculite) 

were also tested. When appropriate, light was reduced 

from the rooting zone by covering the bases of vessels with 

aluminum foil. In all cases, shoots were transferred to the respective 

auxin-free support system for a 90-day culture period. 

In a second series of experiments, the effects of the addition 

of several ancillary compounds to media containing IBA or 

NAA (0, 6 and 12 µM) were tested: (1) 0.25 mg l –1 paclobutrazol, 

an inhibitor of gibberellin biosynthesis; (2) 3 or 

6mMCa 2+ (as calcium gluconate); (3) 10 µM coumarin; (4) 2, 

5, 10 and 20 µg l –1 triacontanol; (5) 1, 3, 5, 7 and 10 mg l –1 

pectimorf; (6) 15 and 60 g l –1 sucrose; (7) 150 or 300 mg l –1 

H3PO4 and 500 mg l –1 glucose; and (8) 0.5 and 1% conifer-derived 

activated charcoal. Shoots were cultured for 30 days in 

their respective media and subsequently transferred to basal 

medium for 60 days. Cultures including coumarin were kept at 

26 and 20 °C. 

In a last experiment, shoots were infected with Agrobacterium 

rhizogenes LBA9402 strain. Basal shoot infection was 

performed either by dipping the basal end of the explants directly 

into the bacterial suspension or after the shoot had been 

wounded with a blade. In some cases, the bacterial suspension 

was injected into the basal cut surface of the shoots. After infection, 

explants were blot-dried between sterile filter paper 

and placed on medium with or without auxins (10 µM IAA or 

IBA), and incubated at 28 ± 1 °C in darkness for 24 h. Subsequently, 

the explants were transferred to medium containing 

200 mg l –1 cefotaxime. 

In all experiments, rooting percentage and the number of 

roots per rooted shoot were recorded. Each treatment contained 

10–12 replicates. 

Statistical analysis 

Significance of treatment effects was assessed by analysis of 

variance employing a completely random design. Percentage 

data were subjected to arcsine transformation before analysis. 

Variation among treatment means was analyzed by Tukey’s 

(1953) procedure. All experiments were conducted at least 




twice. A hierarchic analysis of variance (nested ANOVA, 

Sokal and Rohlf 1995) design was also used to estimate variance 

components for the morphogenic parameters recorded, 

partitioning the variation among seed lots and among seedlings 

within lots. All analyses were performed with the Super- 

ANOVA program (Abacus Concepts, Berkeley, CA). 

Results 

Axillary shoot proliferation from juvenile explants 

Nutrient medium, growth regulators and agar brands Fewer 

than 5% of apical and nodal explants of C. atlantica and C. libani 

sprouted during 40 days of culture, although most explants 

formed a callus. The frequency of sprouting was unaffected 

by pulse treatments with BA or TDZ , nutrient medium 

(MSH, MBF or MSBN/2), the presence of BA in the culture 

medium or the agar brand (Difco-Bacto, Phytagel, Pronadisa 

or A-1296) (data not shown). Agar A-1296, which has been 

used previously for C. libani cultures (Piola and Rohr 1996), 

was selected for subsequent experiments. Maximum callus formation 

(80–90%) was observed when apical explants were 

first dipped in BA or TDZ solution and then transferred to 

BA-supplemented medium. Although some calli underwent 

necrosis, others differentiated adventitious buds and needles. 

These organogenic responses were mainly observed in apical 

explants from C. atlantica and C. libani growing in the presence 

of 9 µM BA. Organogenic calli were subcultured on their 

respective basal media with or without activated charcoal at either 

26 °C or at 30 °C (Piola and Rohr 1996), but the treatments 

were without significant effect on bud elongation. 

Explant size, temperature and growth regulators Shoot apices 

and microcuttings, isolated from 3-month-old C. libani and 

C. atlantica seedlings, were cultured at 30 °C on MSBN/2 medium 

with or without 9 µM BA. Most of the shoot apices grown 

in the presence of BA produced a callus. Culture survival percentages 

(85–100%) were not significantly affected by explant 

size in either species; however, axillary bud proliferation oc- 


curred only when microcuttings were used as primary explants 

(Figure 1). In both species, sprouting percentages and mean 

number of axillary shoots per explant were significantly increased 

in microcuttings grown on medium without BA (80% 

without BA versus 60% with BA, P = 0.05; 3.0 versus 1.5, P = 

0.05). 

In another experiment, entire or decapitated C. libani and 

C. atlantica microcuttings were cultured on MSBN/2 medium 

with or without BA or Z and kept for 45 days at 30 or 26 °C. In 

C. libani cultures, survival and sprouting percentages ranged 

from 85 to 100%, and these responses were not significantly 

affected by incubation temperature or the BA and Z treatments 

(P > 0.05) (data not shown). However, decapitation of C. libani 

microcuttings significantly reduced the mean number of 

shoots formed per explant (3.0 versus 4.1, P = 0.05; Table 1). 

Basal MSBN/2 medium or Z was more effective than basal 

medium + BA in promoting axillary shoot proliferation from 

cultured C. libani microcuttings (4.0 or 4.5 versus 2.1 shoots 

per explant, respectively; P = 0.05, Table 1). 

In C. atlantica cultures (Table 2), an incubation temperature 

of 30 °C and explant decapitation both reduced microcutting 

survival percentages (69.4 versus 87.8%, P = 0.05; 87.8 versus 

93.1%, P = 0.05; respectively). Incubation temperature, decapitation 

and growth regulators all significantly influenced 

sprouting percentages, the best results being obtained at 26 °C 

(82.3 versus 66.7%, P = 0.05), with entire explants (84.9 versus 

64.1%, P = 0.05) and basal or Z-supplemented medium 

(83.8 or 77.1% versus 62.7%, P = 0.05). The detrimental effect 

of incubation at 30 °C versus at 26 °C was particularly evident 

in decapitated microcuttings. The mean number of axillary 

shoots formed per cultured explant was higher in entire microcuttings 

(2.6 versus 1.3, P = 0.05) and the presence of BA reduced 

this response (1.2 versus 2.4 and 2.1 shoots per explant 

in Z-supplemented medium or basal medium, respectively; 

P = 0.05). Significant interactions between explant type and 

incubation temperature or presence of cytokinin in the medium 

were also evident, with the highest mean number of 

shoots per explant obtained when entire microcuttings were 

Figure 1. Axillary shoot proliferation 

from C. atlantica (A) and C. libani (B) 

microcuttings cultured on MSBN/2 

without growth regulators. 



Table 1. Effects of temperature, explant type and cytokinins on shoot 

proliferation from microcutting cultures of juvenile C. libani. Values 

are combined means from two experiments of 10 observations each. 

Cultures were established on MSBN/2 medium and culture time was 

60 days. Values followed by different letters are significantly different 

according to Tukey’s test at P < 0.05. 

Microcutting Temperature Shoots per explant 

(°C) Cytokinin (µM) 

cultured at 30 °C in either basal medium or Z-supplemented 

medium (Table 2). 

Based on these results, entire microcuttings of C. atlantica 

and C. libani were cultured at both 26 and 30 °C in the presence 

of Z or TDZ (Table 3). In both species, survival percentages 

were lower when microcuttings grown on cytokinin- supplemented 

medium were kept at 30 °C. This negative effect of 

the higher temperature was especially evident in C. atlantica 

microcuttings cultured in the presence of TDZ (40%). Sprouting 

percentages ranged from 80 to 100% and neither incubation 

temperature nor cytokinin type significantly influenced 

this response (P > 0.05, data not shown). In contrast, mean 

number of shoots formed per explant was higher at 30 °C than 

at 26 °C (4.1 versus 2.9, P = 0.05), and Z was generally more 

effective than TDZ (4.1 versus 2.7, P = 0.05). Under the culture 

conditions tested, shoot proliferation rates were higher in 

C. libani than in C. atlantica microcuttings (4.7 versus 2.4, P = 


0.0 2.2 BA 2.2 Z Mean 1 

Entire 26 4.4 2.3 5.3 

30 5.3 3.1 3.9 

Decapitated 26 3.6 1.3 4.6 

30 2.8 1.6 4.1 

Mean 2 

1 Effect of explant type. 

2 Effect of cytokinin type. 

4.0 a 2.1 b 4.5 a 

4.1 a 

3.0 b 


0.05). The maximum number of shoots formed per explant 

(7.0) was obtained when C. libani microcuttings were cultured 

on basal medium at 30 °C. This temperature effect was less evident 

in microcuttings isolated from shoot-proliferating cultures. 

In all experiments, and irrespective of the treatments, the 

sprouted buds reached a final length of 1 to 2 cm. 

These experiments were performed with seedlings from different 

seed stocks obtained from two commercial companies. 

To determine the variance attributable to the seed lots and to 

the individuals within each lot, the results were subjected to a 

nested ANOVA, which indicated that there was no significant 

effect of seed lot on survival or sprouting percentage. In contrast, 

variations in shoot yield were due to differences among 

seed lots (23 and 35% in C. atlantica and C. libani, respectively) 

and among seedlings within seed lots (77 and 65% for 

C. atlantica and C. libani, respectively). 

Axillary shoot proliferation from explants of mature origin 

None of the culture conditions tested promoted axillary shoot 

proliferation from apical shoot meristems of mature trees of 

C. atlantica or C. libani. Most of the responding explants 

(60–70%) formed calli. Neither the culture medium (SH or 

MS/2) nor growth regulators (BA or NAA, or both) affected 

this morphogenic response. Meristem-derived calli were maintained 

in culture for almost 6 months, but attempts to induce 

organogenesis failed. 

Based on the data obtained with seedlings, shoot apices 

(0.6 cm) from 30-year-old C. libani were surface sterilized 

with either NaClO or HgCl2 and cultured on MSBN/2 medium 

with or without BA or NAA or both. Although the effectiveness 

of the two sterilizing agents was similar (an 80% yield of 

axenic explants), NaClO caused generalized chlorosis in some 

explants. Survival percentage was 50% and neither the sterilizing 

agent nor the presence of growth regulators significantly 

affected this result. Explant elongation and subsequent axillary 

bud sprouting was observed only in cultures established 

on basal medium. Under these conditions, 15% of the explants 

elongated and showed bud sprouting (1–3 buds per explant) 

within 2–3 months of culture. Explants grown in the presence 

of BA with or without NAA formed calli. 

Table 2. Effects of temperature, explant type and cytokinins on shoot proliferation from microcutting cultures of juvenile C. atlantica. Values are 

combined means from two different experiments of 10 observations each. Cultures were established on MSBN/2 medium and culture time was 

60 days. 

Microcutting Cytokinin (µM) Survival (%) Sprouting (%) Shoots per explant Mean 1 

26 °C 30 °C 26 °C 30 °C 26 °C 30 °C 

Entire 0.0 100.0 100.0 92.3 91.7 1.9 3.8 2.9 a 

2.2 BA 100.0 83.3 66.7 75.0 1.1 1.6 1.4 b 

2.2 Z 91.7 83.3 91.7 91.7 2.5 4.4 3.5 a 

Mean 2 

83.6 a 86.2 a 1.8 b 3.3 a 

Decapitated 0.0 76.9 41.7 84.6 66.7 1.3 1.4 1.4 b 

2.2 BA 83.3 75.0 75.0 33.3 1.8 0.4 1.1 b 

2.2 Z 75.0 33.3 83.3 41.7 1.5 1.2 1.4 b 

Mean 2 

81.0 a 47.2 b 1.5 b 1.0 b 

1,2 

Interaction of explant type with cytokinins or temperature, respectively. For each interaction, values followed by different letters are significantly 

different according to Tukey’s test at P ≤ 0.05. 




Table 3. Effects of temperature and cytokinins on shoot proliferation from microcutting cultures of juvenile C. libani and C. atlantica. Values are 

combined means from two experiments of 12–14 observations each. Cultures were established on MSBN/2 medium and culture time was 60 days. 

Within a column, values followed by different letters are significantly different according to Tukey’s test at P ≤ 0.05. 

Species Temperature (°C) Cytokinin (µM) Survival (%) Shoots per explant 

C. libani 26 0.0 90 ab 4.2 bcde 

2.2 Z 80 ab 4.8 ab 

0.45 TDZ 90 ab 3.0 bcdef 

30 0.0 90 ab 7.0 a 

2.2 Z 60 bc 4.6 abc 

0.45 TDZ 80 ab 4.5 abcd 

C. atlantica 26 0.0 100 a 1.7 ef 

2.2 Z 100 a 2.7 bcdef 

0.45 TDZ 100 a 1.5 e 

30 0.0 100 a 2.0 def 

0.45 TDZ 40 c 2.1 cdef 

Under the most favorable conditions of those tested (sterilization 

with HgCl2 and culture on basal MSBN/2 medium), the 

percentage of microcuttings from 10–15-year-old C. libani 

showing axillary bud sprouting ranged from 15 to 20%. To obtain 

enough material for further experiments, sprouted buds 

were routinely isolated and subcultured on basal MSBN/2 medium. 

Excised shoots from these proliferating cultures were 

used to study the effects of nutrient medium (WPM and 

MSBN/2) and cytokinin type (TDZ and Z) on axillary bud proliferation 

(Table 4). Survival percentages ranged from 90 to 

100%, except when microcuttings were cultured on TDZ-supplemented 

MSBN/2 medium (survival = 60%), as found for 

explants of juvenile origin. Both nutrient medium and cytokinin 

type significantly influenced sprouting percentage and 

mean number of axillary buds formed per explant and there 

was a significant interaction between these factors. In cultures 

established on MSBN/2, all microcuttings showed axillary 

bud sprouting and the mean number of developed buds per 

explant (3.7–6.2) was not significantly influenced by the presence 

and type of cytokinin. In contrast, cytokinin enhanced 

axillary bud proliferation in microcuttings grown on WPM, 

the best results (100% sprouting and 7.3 buds per explant) being 

obtained in the presence of Z. 


Although initial multiplication rates were low, they increased 

during the first few subcultures, providing enough material 

for mass multiplication of adult C. libani. When this protocol 

was used for the in vitro propagation of 200-year-old 

C. libani trees, we achieved successful in vitro establishment 

in three out of the six sampled trees (genotypes AL1, AL2 and 

AL3), suggesting that in vitro establishment was genotype-dependent. 

The initial multiplication efficiency of the three 

genotypes (as measured by the frequency of axillary bud breaking 

after 2 months of culture) was 3.0, 7.6 and 9.3%, respectively. 

Multiplication efficiency of the three genotypes increased 

with number of subcultures, and within 6 months, 

mean sprouting percentage reached 70% for all three genotypes 

and this rate was maintained during subsequent subcultures, 

yielding 20–30 clones of each genotype after 2 years. 

Adventitious bud differentiation from cultured embryos of 

C. atlantica 

Based on a previous report of successful adventitious bud induction 

from cultured leaves of mature Juniperus oxycedrus 

(Gómez and Segura 1994), needles from C. atlantica and 

C. libani seedlings were cultured on MSH medium with 

0.5 µM BA. Although some needles formed a callus in the 

presence of BA (23 and 13% in explants from C. atlantica and 

Table 4. Effects of nutrient media and cytokinins on shoot proliferation from 10–15-year-old C. libani microcuttings. Explants were first cultured 

for 60 days on media with or without cytokinin and then transferred to their respective basal media. Values are combined means from two different 

experiments of 10 observations each and culture time was 90 days. Within a column, values followed by different letters are significantly different 

according to Tukey’s test at P ≤ 0.05. 

Nutrient medium Cytokinin (µM) Survival (%) Sprouting (%) Shoots per explant 

WPM 0 90 a 10 a 0.2 c 

0.45 TDZ 100 a 100 b 3.4 bc 

2.2 Z 100 a 100 b 7.3 a 

MSBN/2 0 100 a 100 b 4.4 b 

0.45 TDZ 60 b 100 b 3.7 b 

2.2 Z 100 a 100 b 6.2 ab 



C. libani, respectively), none of the cultured explants differentiated 

adventitious buds. 

In subsequent experiments, mature C. atlantica embryos 

were cultured on MSBN/2 medium with or without Z and BA. 

Within the first week of culture, embryos elongated and the 

cotyledons and hypocotyls became green. Embryos on basal 

medium produced no callus or adventitious buds, developing 

as normal seedlings. In the presence of cytokinins, hypocotyls 

and cotyledons in contact with the medium proliferated quickly, 

producing callus. Usually the radicles did not show a response 

either in color or in cell proliferation. The first adventitious 

buds were directly induced from the upper surface of the 

hypocotyls (Figure 2A) after 20–30 days of culture. Further 

bud differentiation occurred on the surface of the previously 

induced calli. Indirect needle primordia differentiation was 

also observed, especially when embryos were cultured in the 

presence of BA. After 50–60 days, the entire upper surface of 

the responding embryos was covered with adventitious buds 

and needles, although the latter did not develop into shoots. 

Table 5 summarizes the bud differentiation process in cultured 

C. atlantica embryos. The bud-forming capacity of the 

explants depended on cytokinin type and concentration, the 

best results being obtained when the embryos were cultured in 

the presence of 9 µM Z (47% of caulogenic explants and 

4.2 adventitious buds per embryo), whereas 9 µM BA induced 

a smaller response (13% and 0.3, respectively). Embryo subculture 

to medium with 4.4 mM Z enhanced the development 

of adventitious buds (Figure 2B). Further shoot elongation was 

achieved following excision and transfer of these shoots to 

hormone-free medium. Transfer of embryos bearing adventitious 

buds to basal medium did not promote bud elongation 

(data not shown). 

Although WPM provided a greater percentage of embryos 

with buds than MSBN/2 medium (49.0 versus 40.0%, P = 

0.05), the mean number of buds formed per embryo in the 

presence of 9 µM Z was similar in the two media (3.1). Irrespective 

of the nutrient medium, 18 µM Z enhanced acicular 

primordia differentiation and reduced adventitious bud differentiation. 

Rooting 

Rooting experiments were carried out with axillary and adventitious 

shoots isolated from proliferating cedar cultures of ju- 



Table 5. Effects of cytokinin type and concentration on the differentiation 

of adventitious buds from embryos of C. atlantica. Values are 

combined means of results from two experiments of 20 observations 

each. Cultures were established on MSBN/2 medium and culture time 

was 60 days. Within a column, values followed by different letters are 

significantly different according to Tukey’s test at P ≤ 0.05. 

Cytokinin Concentration Explants with Buds per 

(µM) buds (%) explant 

Z 0.0 0.0 0.0 

2.2 13.3 0.8 

4.4 33.3 1.3 

6.6 33.3 0.8 

9.0 46.7 4.2 

Mean 25.3 a 1.4 a 

BA 0.0 0.0 0.0 

2.2 6.7 0.1 

4.4 6.7 0.1 

6.6 13.3 0.1 

9.0 13.3 0.3 

Mean 8.0 b 0.1 b 

venile and adult origin. None of the experimental variables 

tested promoted rooting. 

Discussion 

We observed in vitro axillary bud proliferation of explants 

from juvenile and adult C. atlantica and C. libani trees. In both 

species, explant size was an important factor affecting axillary 

bud proliferation in in vitro culture. Piola and Rohr (1996) reported 

that axillary and apical buds from in-vitro-grown microcuttings 

of C. libani sprout at 30 °C but not at 24 °C. Subsequently, 

Piola et al. (1998) demonstrated that needle removal, 

but not microcutting decapitation, substituted for the higher 

temperature requirement to break bud dormancy. Abscisic 

acid (ABA) concentrations in microcuttings with dormant 

buds were higher than in microcuttings bearing sprouted buds, 

leading these authors to suggest that ABA accumulation in 

needles caused bud dormancy of C. libani microcuttings at 

24 °C. Therefore, we always used defoliated microcuttings, to 

Figure 2. Induction and development 

of adventitious buds from cultured embryos 

of C. atlantica. Adventitious bud 

differentiation on MSBN/2 medium 

with 9 µM Z (left) and adventitious 

bud development on MSBN/2 medium 

with 4.4 µM Z (right). Bar = 0.3 cm. 




exclude a possible ABA influence in our experiments. The use 

of defoliated microcuttings makes it difficult to compare our 

results with those reported by Piola and Rohr (1996) for juvenile 

C. libani microcuttings; nevertheless, we found that an incubation 

temperature of 30 °C enhanced shoot yield in juvenile 

microcuttings of both C. atlantica and C. libani. Piola and 

Rohr (1996) found that a 3-h pulse with 0.1 mM BA induced 

the highest proliferation rate (4.5 buds per explant) in C. libani 

microcuttings. We observed similar results for C. libani microcuttings 

grown in the presence of 2.2 µM BA; however, BA 

negatively affected bud sprouting in C. atlantica microcuttings. 

Nested ANOVA attributed most of the variability in shoot 

yield from cultured microcuttings of juvenile origin to single 

seedlings (77 and 65% for C. atlantica and C. libani, respectively). 

This strong genotypic effect was even more evident in 

cultured explants of bicentennial C. libani trees, where only 

three out of the six genotypes were successfully established in 

vitro. A similar genotypic effect on the bud-forming capacity 

of explants was observed by Tang et al. (2001). In some conifers, 

variation in morphogenic responses was evident not only 

among embryos from different seed lots but also among embryos 

obtained from controlled crossings (von Arnold and 

Eriksson 1982, 1986, Shen and von Arnold 1982). We observed 

differences in the initial in vitro performance of the 

three genotypes, but these differences decreased with increasing 

number of subcultures. 

Usually, in vitro manipulation of woody plants is more difficult 

with mature explants than with juvenile explants, and 

Cedrus was no exception. A comparison between cedar explants 

of juvenile origin and those of adult origin showed that 

microcuttings were the preferred explant for axillary shoot 

proliferation in juvenile and adult cultures. Although this response 

did not require the presence of cytokinin in the culture 

medium, Z increased axillary shoot yield in explants of mature 

origin. Hormone-free nutrient media have been successfully 

employed for culture establishment of other mature Pinaceae 

including Pseudotsuga menziesii (Mirb.) Franco and Pinus 

lambertiana Dougl. (Gupta and Durzan 1985), although the 

presence of growth regulators favored axillary bud sprouting 

in shoot apices and microcuttings isolated from 20-year-old 

Larix occidentalis Nutt. trees (Chesick et al. 1990). 

Shoot proliferation rates in cultures of adult C. libani were 

generally greater on basal MSBN/2 medium than on basal 

WPM. Although the influence of nutrient medium on in vitro 

morphogenesis is well documented, its effect remains one of 

the most empirical aspects of plant cell culture (Preece 1995). 

Compared with basal MSBN/2 medium, basal WPM has 

higher concentrations of calcium, sucrose, micronutrients and 

organic supplements, and contains biotine and folic acid (both 

absent in MSBN/2 medium). A reduced amount of nutrients 

stimulates axillary branching in some plants (Karhu 1997) and 

low calcium availability can affect apical meristem integrity 

(McCown and Sellmer 1987), thus favoring axillary bud development; 

however, we are unable to offer an explanation for 

the differential effect of MSBN/2 and WPM media on the 

basis of our experimental data. 


Adventitious organogenesis offers higher potential for 

shoot production than axillary bud proliferation, and is the preferred 

method for coniferous micropropagation because coniferous 

buds are generally induced directly on the explant 

(Thorpe et al. 1991). With a few exceptions, however, techniques 

for adventitious budding of coniferous explants obtained 

from trees in the adult growth phase are still limited, because 

the caulogenic potential of isolated organs is affected by 

the ontogenic age of the tissues (von Aderkas and Bonga 2000, 

Giri et al. 2004). In our experiments, callus derived from shoot 

apices differentiated adventitious buds in the presence of cytokinin, 

but only when explants of juvenile origin were used and 

we were unable to promote their elongation. In contrast, 

Hosseyni et al. (1999) reported the induction of adventitious 

organogenesis (buds and needles) from winter buds of 10–15year-old 

C. libani cultured on WPM with BA or kinetin. This 

difference between studies may be associated with tree age, 

because our trees were older. 

We successfully generated adventitious shoots from C. atlantica 

embryos, which opens up the possibility of mass propagation 

of this species. Our regeneration system included: (1) 

adventitious budding in the presence of 9.0 µM Z; (2) bud development 

on medium containing 4.4 µM Z; and (3) shoot 

elongation on cytokinin-free medium. Zeatine or benzyladenine 

alone was enough to stimulate both direct and indirect 

adventitious budding, which is in agreement with previous 

findings in other conifer species (Thorpe et al. 1991); however, 

the presence of Z improved the bud-forming capacity of the 

explants, and the process of bud development required transfer 

of C. atlantica embryos to a medium with cytokinin. In contrast, 

in other conifers, bud development depends on transfer 

of explants to hormone-free medium (Gómez and Segura 

1994, Mata et al. 2001, Villalobos-Amador et al. 2002, Schestibratov 

et al. 2003). 

Successful micropropagation of many woody species is frequently 

limited by their reluctance to form adventitious roots, 

and C. libani and C. atlantica were no exception. In many species, 

rooting can be achieved by adding auxins to the culture 

medium or by using different support substrates (Hartmann et 

al. 1997). Activated charcoal, a high sucrose concentration, 

variations in environmental temperature conditions and light 

reduction in the rooting zone improve auxin-induced rooting 

in many species, including conifers (George 1993). A variety 

of ancillary compounds such as paclobutrazol, phosphoric 

acid, calcium, triacontanol, oligosaccharins and coumarin 

(George 1993, Haissig and Davis 1994, Tantos et al. 2001, 

Nandi et al. 2002) can improve rooting capacity in recalcitrant 

species. In some conifer species, infection with A. rhizogenes 

enhances rhizogenesis (McAfee et al. 1993, Tzfira et al. 1996, 

Mihaljevic et al. 1998, Villalobos-Amador et al. 2002). In our 

experiments, all of these variables failed to induce rooting in 

shoots of axillary or adventitious origin of C. libani and C. atlantica. 

Nicholson (1984) and Nandi et al. (2002) were able to 

induce rooting in C. deodara (D. Don) G. Don and rooting has 

been reported in C. libani and C. atlantica (Dirr and Heuser 

1987) cuttings, although in the last two species, rooting percentages 

were minimal and rooting was observed only when 



cuttings were sampled at the end of the winter season. Hosseyni 

et al. (1999) also reported rooting (29–33%) from adventitious 

shoots of C. libani cultured on MS/4 or WPM/2 

media with 1.4 µM IAA but not with 1.4 µM IBA; both hormones, 

however, were ineffective in promoting rooting in 

shoots generated from winter buds of 10–15-year-old C. libani 

trees. 

In conclusion, we have developed an efficient method to 

promote axillary bud proliferation from cedar microcuttings 

of juvenile and adult origin. Our regeneration system (bud 

sprouting in defoliated microcuttings cultured on basal MSBN/2 

medium at 26 or 30 °C, and isolation and continuous subculture 

of sprouted buds onto the same medium) provides a means 

of obtaining multiple shoots from a single microcutting from 

cedar trees old enough to have demonstrated their superior 

characteristics. Our regeneration method may be useful for 

clonal propagation of elite genotypes provided that a protocol 

for the successful rooting of proliferating shoots is developed. 

The phenotypic characteristics of the putative clones were 

similar to those of seedlings grown from seeds. We also developed 

a protocol for the successful regeneration of adventitious 

shoots from mature embryos of C. atlantica. 

Acknowledgments 

The authors thank the European Union, Contract Number ERBIC18- 

CT97-0177, and Generalitat Valenciana (Grupos 03/102) for financial 

support. 

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