American Journal of Plant Sciences, 2011, 2, 237-244
doi:10.4236/ajps.2011.22025 Published Online June 2011 (http://www.SciRP.org/journal/ajps)
Direct Organogenesis from Cotyledons in
Cultivars of Citrus clementina Hort. Ex Tan
Giovanna Lombardo1, Roberta Alessandro2, Anna Scialabba1, Mariangela Sciandra3,
Fabio De Pasquale4
1Department of Environmental Biology and Biodiversity, University of Palermo, Palermo, Italy; 2Department of Agro and Environ-
mental Systems (S.Ag.A.), University of Palermo, Palermo, Italy; 3Department of Mathematical and Statistical Sciences “Silvio
Vianelli”, University of Palermo, Palermo, Italy; 4Institute of Plant Genetics, National Council of Research, Research Division Pal-
ermo, Palermo, Italy.
Received March 1st, 2011; revised April 23rd, 2011; accepted April 30th, 2011.
ABSTRACT
An efficient protocol to induce shoot buds regeneration in Citrus clementina cultivars (“Monreal”, “SRA 63” and “SRA
64”) by direct organogenesis has been developed using cotyledons as explants. Cotyledons transversely cut in three
segments and entire ones were cultured on Murashige and Skoog (1962) solidified medium containing vitamins, 500
mg·l−1 malt extract, 50 g·l−1 sucrose and supplemented with three different concentrations of BAP (8.8, 13.2 and 17.6
μM). In all three cultivars the entire cotyledons showed more shoot morphogenic potential than transversely cut ones
and after 60 incubation days the optimum BAP concentration was 17.6 μM in “Monreal” (50% ± 2.89% of frequency
regeneration) and 13.2 μM in “SRA 63” (33.33% ± 3.33%) and “SRA 64” (25.93% ± 1.85%). In absence of BAP No
morphogenesis occurred, demonstrating the absolute requirement of this hormone for shoots induction. The young
shoots showed a regular growth in the culture tubes containing the basal medium without hormones, and the rooted
plantlets survived after acclimatization. This protocol may find application in Citrus genetic improvement programs.
Keywords: Citrus clementina, Cotyledons, Direct Organogenesis, Plant Regeneration, Tissue Culture
1. Introduction
Citrus clementina Hort. ex Tanaka is a very important
crop because of its excellent organoleptic and easy-
peeling qualities. In C. clementina, as in Citrus spp., bud
mutations arise spontaneously [1-4] and growers select
them to improve horticultural traits of economically in-
teresting species; there is also a high degree of inter-
specific sexual compatibility, so clementine is highly
used in genetic improvement programs as mother plant to
obtain new genotypes through hybridization by con-
trolled inbreeding.
In vitro technique is a useful method to obtain true-
to-type regenerated plants [5,6] as well as to induce so-
matic mutations [7] affecting characters of agronomic
interest in order to develop new and improved genotype
lines. There are several studies on plant regeneration by
organogenesis and embryogenesis from different types of
explants in Citrus genus [8-13], but very little informa-
tion is available about procedures for achieving regen-
eration from clementine mandarin: embryos were in-
duced from calli of ovular tissue [14], aberrant plants
were obtained by direct and indirect somatic embryo-
genesis from the nucellus of eight cultivars [7], and gy-
nogenesis [15] and androgenesis [16-18] were studied in
different cultivars.
Cotyledons have high potential of regeneration [19,20]
and represent a good source of tissue cultures. Organo-
genesis from cotyledons was successfully obtained in
Pongamia pinnata (L.) [21], Glicine max (L.) Merril [22],
Dalbergia sissoo Roxb. [23], Capsicum annum L. [24]
and Citrullus lanatus (Thunb.) Matsum. & Nakai [25].
Beyond, in the last decade cotyledons and cotyledonary
nodal regions were used as target tissues for transforma-
tion mediated by Agrobacterium [20].
The aim of this work was to induce in vitro plant re-
generation in Citrus clementina to be used in Citrus ge-
netic improvement programs. Three cultivars were inves-
tigated in the experiments and cotyledons were used as
explants.
2. Materials and Methods
Ripe fruits of C. clementina “Monreal”, “SRA 63” and

Direct Organogenesis from Cotyledons in Cultivars of Citrus clementina Hort. Ex Tan 238
“SRA 64” of the germplasm collection belonging to the
Istituto di Genetica Vegetale (sezione di Palermo), Na-
tional Research Council (CNR) of Italy, were sampled at
the beginning of December. The seeds, derived from
open pollination, were drawn aseptically from the fruits
into a laminar air flow and deprived of two teguments
and of the embryo axis with a scalpel and forceps. Entire
cotyledons (EC) and transversely cut cotyledons (CC)
were used as explants. EC has one cut surface only,
while CC were obtained cutting the cotyledon into three
segments: the segment close to the embryo axis, identi-
fied as proximal (CCp), and the one next to it, called
middle (CCm), have two cut sides each, while the far-
thest segment is the distal (CCd) and has one cut side.
EC and CC were cultured on Murashige and Skoog [26]
solidified medium containing vitamins, 500 mg·l−1 malt
extract and 50 g·l−1 sucrose as basal medium. Three dif-
ferent concentrations of 6-benzylaminopurine (BAP)
were tested in the basal medium: 8.8 μM, 13.2 μM and
17.6 μM. The pH of the medium was adjusted to 5.7 with
1 N KOH and the medium was autoclaved at 103 kPa
(121˚C) for 20 min. Shoots about 5 mm long were iso-
lated from cotyledons and transferred into culture tubes
containing a hormone-free medium. Preliminary experi-
ments were performed in the same culture conditions
using seeds of fruits collected in the middle of November.
All explants were incubated in a climate chamber at 26˚C
± 1˚C under 16-h photoperiod. Rooted plants were trans-
ferred into Jiffy 7® peat pellets to the green house and,
when the radical apparatus was formed, they were potted
and slowly submitted to acclimatization. Experiments
were performed with 10 seeds per treatment and repeated
thrice. The regeneration frequency (RF) (number of ex-
plants producing buds or shoots per total number of ex-
plants cultured multiplied 100), the number of
buds/shoots per explant and shoot elongation were meas-
ured at 30, 60 and 90 days of culture.
Statistical Analysis
The statistical analysis was carried out at first through
a descriptive analysis in which the data were presented as
a mean value with its relative standard error by using
Microsoft Office Excel 2003.
Following, the distributions of regeneration frequency,
number of buds/shoots per explant and shoot elongation
have been studied in order to properly specify a statistical
model able to relate these dependent variables to the
three explanatory variables BAP concentration, explant
type and genotype.
BAP concentration was considered as a continuous
variable while explant type and genotype were consid-
ered as factor variables with respectively four (EC, CCd,
CCm, CCp) and three (“Monreal”, “SRA 63”, “SRA 64”)
levels.
The relationship between dependent and explanatory
variables was modelled by using three different General-
ized Linear Models (GLM) [27] according to the distri-
bution of the response variables considered. In particular,
in the regeneration frequency case, being a dichotomous
variable (0 = No regeneration, 1 = regeneration), a bino-
mial GLM was fitted; number of buds/shoots per explant
was modelled through a Poisson GLM as it is a counting
variable and finally shoot elongation was modelled by a
Gamma GLM as its distribution showed a marked posi-
tive skewness. The best three GLMs resulted from a
model selection procedure based on the Akaike Informa-
tion Criterion (AIC) [28].
The statistical modelling has been carried out using R
(R Development Core Team 2005) [29], a public domain
statistical environment freely downloadable from the
URL www.R-project.org.
3. Results and Discussion
Shoots began to emerge directly from the explants after
three weeks of incubation and no callus around the
emerging shoots was observed under the stereo micro-
scope (Figure 1(a)). Callusing alone occurred very rarely
and indirect organogenesis was never observed. The
shoots in formation appeared as clusters of green protu-
berances that successively differentiated into buds (Fig-
ure 1(b)). They arose more frequently from the cut sides
(Figure 1(c)) in both EC and CC and rarely directly from
the intact cotyledon surface. Regeneration in all geno-
types only occurred in the presence of BAP (Figure 2),
that has a significant effect (p ≤ 0.001) on the regenera-
tion probability (Table 1(A)), while neither swelling nor
morphogenic responses were noted when using the hor-
mone-free basal medium. All BAP concentrations in-
duced morphogenesis in “Monreal” and “SRA 64”,
whereas in “SRA 63” only the lowest level of BAP (8.8
μM) in EC and the highest concentration (17.6 μM) in
the proximal segments were not inductive after 90 cul-
ture days (Figure 2). The young differentiated shoots
transferred to the basal medium without growth regula-
tors rooted easily and put new leaves, showing a regular
growth (Figure 1(d)). The plantlets survived to the
transfer to Jiffy 7® peat pellets into the green house and
were acclimatized successfully after having been potted
(Figure 1(e)). The entire process, from shoot emergence
to plant acclimatation, was accomplished approximately
in four-six months.
“Monreal” (Figure 2(a)) was the most reactive and
fastest cultivar in terms of RF but only for EC explants:
after 30 incubation days the RF was 33.33 ± 2.22 and
41.48 ± 1.48 respectively in 13.2 μM and 17.6 μM of BAP
and an increasing trend to raise hormone concentration

Direct Organogenesis from Cotyledons in Cultivars of Citrus clementina Hort. Ex Tan 239
was observed. The best regenerative potential was ob-
tained by EC in 17.6 μM (50% ± 2.89% RF) after 60
incubation days. Proximal, middle and distal segments
morphogenically responded in an unremarkably different
way to BAP treatment.
In “SRA 63” (Figure 2(b)) the best BAP concentra-
tion for all explants was 13.2 μM and the most regenera-
tive were EC and the middle segments (in both 33.33% ±
3.33% RF); these regeneration percentages were reached
after 60 incubation days and held steady in both explants
after 90 days. A slightly lower regenerative response was
obtained in the distal segments in 13.2 μM of BAP
(26.67% ± 3.33%), while 17.6 μM concentration induced
poor organogenesis, ranging from 6.67% ± 1.67% (distal
segment) to 13.33 ± 3.33 (EC and middle segment) after
90 days of incubation.
In “SRA 64” (Figure 2(c)) the best regeneration oc-
curred in EC in BAP 13.2 μM (30.04% ± 3.92% RF)
after 90 incubation days. Regarding CC, the best morpho-
genetic response was obtained in the distal segments in
BAP 13.2 μM (21.85 ± 0.37 RF) and in the middle seg-
ments in both 8.8 and 13.2 μM (18.52 ± 3.70) concentra-
tions after 90 incubation days. Regeneration was very
low in proximal segments, ranging from 3.70 ± 1.85
(BAP 17.6 μM) to 7.04 ± 1.48 (BAP 13.2 μM).
The probability of regeneration (Table 1(A)) is dif-
ferent in the three genotypes: “SRA 64” (p_0.001) had a
probability of regeneration lower than the “Monreal”,
while the probability of regeneration for the “SRA 63”
(p_0.05) resulted marginally different from the “Monreal”.
Beyond, the probability of regeneration for the entire
cotyledon was significantly higher than the other three
explant types (Table 1(A)).
The number of buds/shoots differentiated per explant
after 60 days of incubation (Table 2) ranged from 1 to
4.28 ± 1.37 in all cultivars. Only the explant types re-
sulted significant (Table 1(B)) so the other two variables
(BAP concentration and genotype) were eliminated from
the model in the Table 1(B). Significative differences in
the number of buds/shoots per explant variable were
achieved between the entire cotyledon and the distal (p ≤
0.001) and medium (p ≤ 0.05) segments; on the contrary
entire cotyledon and proximal segment are not significa-
tively different between them and they were the explants
with the highest number of buds/shoots differentiated per
explant.
The shoots generated from EC explants (Table 2) was
always significatively much longer (p ≤ 0.001) than the
Table 1. Akaike Information Criterion statistical procedure on the regeneration frequency variable (A), the number of
buds/shoots per explant variable (B) and the shoot elongation variable (C) in Citrus clementina cultivars, estimated respec-
tively from a GLM-binomial, GLM-Poisson and GLMGamma (link Identity). Intercept 1 represents “Monreal”, hor-
mone-free medium and entire cotyledon; intercept 2 and 3 represent entire cotyledon; 6-benzylaminopurine (BAP); trans-
versely cut cotyledons distal (CCd), middle (CCm) and proximal (CCp) segments. Significance codes: ‘***’ p ≤ 0.001; ‘**’ p ≤
0.01; “*” p ≤ 0.05; “.” p ≤ 0.1; “ ” p ≤ 1. Standard error (SE).
Estimate SE z- p-value
Intercept 1 –2.366 0.296 –7.988 1.37e -15
BAP 0.113 0.018 6.129 8.82e-10
“SRA 63” –0.453 0.247 –1.830 0.067
“SRA 64” –0.960 0.228 –4.214 2.50e-05
CCd –0.728 0.257 –2.827 0.004**
CCm –0.574 0.249 –2.304 0.021*
A Regeneration
Frequency variable
CCp –1.047 0.278 –3.763 0.0001***
Intercept 2 1.247 0.074 16.780 <2e-16***
CCd –0.505 0.146 –3.454 0.0005***
CCm –0.324 0.132 –2.451 0.014*
B
Number of
buds/shoots per
explant variable
CCp 0.005 0.136 0.040 0.967
Intercept 3 5.117 0.560 9.131 9.68e-16
CCd –2.394 0.684 –3.498 0.0006***
CCm –3.1549 0.621 –5.082 1.24e-06***
C Shoot elongation
variable
CCp –2.612 0.701 –3.725 0.0002***

Direct Organogenesis from Cotyledons in Cultivars of Citrus clementina Hort. Ex Tan 240
(a) (b)
(c)
(d) (e)
Figure 1. Different development stages in the plantlet formation via direct organogenesis from cotyledons in Citrus clementina
“Monreal”; the same pattern occurred in “SRA 63” and “SRA 64”. (a) Regeneration appeared with clusters of green swelling
and protuberances (arrows); (b) Differentiation of bud (arrow); (c) Shoots arising from the wounded side; (d) Shoot trans-
ferred in the hormone-free basal medium showing a regular growth; (e) Potted plant established in vivo conditions. Bars = 1
mm.

Direct Organogenesis from Cotyledons in Cultivars of Citrus clementina Hort. Ex Tan241
Figure 2. Effect of BAP concentration on regeneration frequency (%) from entire and transversely cut cotyledons (proximal,
middle and distal segments) of Citrus clementina cultivars, “Monreal” (a) “SRA 63” (b) and “SRA 64” (c), at 30, 60 and 90
days of incubation. Vertical bars represent standard error of the means.

Direct Organogenesis from Cotyledons in Cultivars of Citrus clementina Hort. Ex Tan 242
Table 2. Influence of BAP concentration on number of buds/shoots differentiated per explant by direct organogenesis and the
shoots’ length (mm) after 60 days of incubation in “Monreal”, “SRA 63” and “SRA 64” Citrus clementina cultivars, in entire
cotyledons (EC) and transversely cut cotyledons (CC), proximal (p), middle (m) and distal (d) segments. Mean ± Standard
error.
Number of Buds/Shoots per Explant Length of Shoots
CC CC
Clementine
Cultivars
BAP
(μM) EC
p m d
EC
p m d
8.8 3.14 ± 0.791.66 ± 0.33 2.75 ± 1.102.66 ± 0.913.82 ± 0.882.66±0.66 2.50 ± 1.19 2.20 ± 0.35
13.2 4.27 ± 1.184.28 ± 1.37 2.50 ± 0.262.50 ± 0.955.20 ± 1.162.68±0.60 2.38 ± 0.57 3.75 ± 1.86 “Monreal”
17.6 3.6 ± 0.68 3.25 ± 1.10 2.40 ± 0.602 ± 0.50 5.98 ± 1.243.95±2.05 1.60 ± 0. 40 1 ± 0
8.8 0 7 3.50 ± 1.501 ± 0 − 2 1.25 ± 0.25 1.33 ± 0.33
13.2 4.2 ± 1.11 2.66 ± 0.33 3.20 ± 0.912.25 ± 0.626.44 ± 2.342±0 2.20 ± 0.84 2.62 ± 1.46
“SRA 63”
17.6 4 0 1 1 7 − 2 ± 0 7
8.8 2.5 ± 0.5 3.50 ± 1.50 2.50 ± 1.501 2 ± 0 1 ± 0 1.90 ± 0.1 4
13.2 3.28 ± 0.713.50 ± 1.50 2.50 ± 0.862 ± 0.77 4.44 ± 1.021.25 ± 0.25 1.75 ± 0.47 3.3 ± 0.7 “SRA 64”
17.6 1.25 ± 0.250 1.66 ± 0.661 4.5 ± 2.02 − 1.33 ± 0.33 1
three types of CC ones (Table 1(C)) and, beyond, the
shoot elongation was not influenced significantly by the
BAP concentration and the genotype. The transfer of
longer shoots to fresh medium has more survival possi-
bility than shorter ones (data not shown).
In previous and preliminary experiments cotyledons of
seeds coming from fruits collected in November showed
a similar pattern of morphogenic response with reference
to the influence of BAP in comparison to seeds of De-
cember: 17.6 μM BAP for “Monreal” and 13.2 μM BAP
for “SRA 63” and “SRA 64” were the best concentra-
tions. Instead the regeneration frequency was higher
(80% RF in “Monreal”, 75% RF in “SRA 63” and 60%
in “SRA 64”) and occurred better in CC than EC. This
behavior may be explained by the fact that cells belong-
ing to juvenile plant material have a higher regeneration
competence and more rapid rates of proliferation in tis-
sue cultures if compared to explants collected from ma-
ture tissue [8,30]. It should be noted that the regeneration
frequency related to the explant physiological state de-
pends on the species (species-specific), as indeed the
mature cotyledons in soybean (Glycine max (L.) Merr.),
for example, were observed to be more regenerative than
immature ones [20]. The morphogenic inducting effect of
wounds [31] may be more productive in younger tissues,
making the CC explants more regenerative than EC.
In the present study, it was demonstrated that BAP is
absolutely required to induce in vitro morphogenesis in
clementine. In Citrus, the cytokinin 6-benzylaminopurine
has been reported in a large number of protocols as pro-
moting the formation of adventitious buds or shoots
[10,32-34]. Its use could reduce the risk of somaclonal
variability in regenerants as opposed to diphenylurea
derivates like N-(2-chloro-4-pyridyl)-N’-phenylurea that
induces high levels of somaclonal variability [35]. The
frequency of regeneration increased with the permanence
in the medium supplemented with BAP, demonstrating
that cells maintained their organogenic competence dur-
ing the 90 incubation days.
Regeneration from cotyledon explants has been re-
ported in several taxa [21-24,36,37] but it has been re-
corded in only a few species belonging to Citrus genus:
adventive embryos formation in Citrus reticulata Blanco
(Nagpur mandarin) and C. jambhiri Lush. (Rough lemon)
[5], indirect somatic embryogenesis in C. reticulata “Lo-
cal Sangtra” [38] and indirect shoot regeneration in C.
grandis (L.) Osbeck (pummelo) [32,39].
4. Conclusions
Shoot regeneration in Citrus clementina can be obtained
through direct organogenesis using cotyledons as explant
and 6-benzylaminopurine growth regulator is essential to
induce differentiation. The best BAP concentration for
inducing regeneration has been determined as 17.6 μM
BAP for “Monreal” and 13.2 μM BAP for “SRA 63” and
“SRA 64” cultivars. The entire cotyledons were always
more regenerative than transversely cut cotyledons for all
the three cultivars, but a different morphogenic response
was observed among the tested genotypes, showing
“Monreal” having the highest organogenic potential.
This protocol may find application in Citrus genetic
improvement and in studies concerning the achievement

Direct Organogenesis from Cotyledons in Cultivars of Citrus clementina Hort. Ex Tan243
of new hybrids reducing the propagation time in respect
to conventional methods. The obtained progeny, hybrid
and heterogeneous, can be multiplied through direct ad-
ventitious shoot organogenesis, thus obtaining more
identical individuals per seed who are more genetically
stable in comparison to plants regenerated via callus, in
which the presence of somaclonal variability is more
probable. The plantlets, that can be transferred success-
fully in vivo in the greenhouse, may be monitored and
evaluated for all new agronomic characteristics.
5. Acknowledgements
This work was supported by Regione Sicilia (Italy).
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