Direct OrgaNogenesis from Cotyledons in Cultivars of Citrus clementina Hort. Ex Tan

doi:10.4236/ajps.2011.22025

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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.

Email: giolom[email protected]

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*

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

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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