The Journal of Horticultural Science and Biotechnology

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A simple protocol for somatic embryogenesis in

Rosa hybrida L. cv. Apollo

Pejman Azadi, Ebrahim Beyrami Zadeh & Valentaine Otang Ntui

To cite this article: Pejman Azadi, Ebrahim Beyrami Zadeh & Valentaine Otang Ntui (2013)
A simple protocol for somatic embryogenesis in Rosa hybrida L. cv. Apollo, The Journal of
Horticultural Science and Biotechnology, 88:4, 399-402

To link to this article: http://dx.doi.org/10.1080/14620316.2013.11512982

Published online: 07 Nov 2015.

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 Journal of Horticultural Science & Biotechnology (2013) 88 (4) 399–402

A simple protocol for somatic embryogenesis in Rosa hybrida L. cv.

Apollo

By PEJMAN AZADI1*, EBRAHIM BEYRAMI ZADEH2 and VALENTAINE OTANG NTUI 3

1
Tissue Culture and Gene Transformation Department, Agricultural Biotechnology Research
Institute of Iran (ABRII), P. O. Box 31535-1897, Karaj, Iran
2
Tissue Culture Department, National Research Center of Ornamental Plants, P. O. Box 37815-137,
Mahallat, Iran
3
Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University,
648 Matsudo, Matsudo, Chiba 271-8510, Japan
(e-mail: azadip@abrii.ac.ir) (Accepted 1 February 2013)

SUMMARY
A successful regeneration system from in vitro-derived leaves of Rosa hybrida cv. Apollo, via somatic embryogenesis,
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has been established. The influence of different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and zeatin on
the induction of embryogenic calli was evaluated. The highest percentage of embryogenic callus induction (48.3%) was
obtained on full-strength MS medium containing 1.0 mg l–1 2,4-D, while at higher concentrations (4.0 or 5.0 mg l–1 2,4-
D) no embryogenic calli were observed. The induction of embryogenic calli also occurred on MS media containing 0.5,
1.0, 2.0, or 4.0 mg l–1 naphthaleneacetic acid (NAA). The lowest concentration of NAA (0.5 mg l–1) showed the highest
rate of production of embryogenic calli. For maturation, embryogenic calli were transferred onto MS media containing
different concentrations of 6-benzyladenine (BA), thidiazuron (TDZ), or abscisic acid (ABA) alone, or in various
combinations. The highest rate of embryo maturation (85.0%) was observed on MS medium supplemented with 1.0 mg
l–1 ABA alone. Mature embryos showed the highest rate of germination on full-strength MS medium containing 2.0 mg
l–1 BA alone. After rooting, well-developed plantlets were maintained under greenhouse conditions. The morphological
characteristics and flower colours of somatic embryo-derived plants were the same as the donor parent.

R ose (Rosa hybrida L.) is the most economically

important ornamental plant.
embryogenesis (SE) offers enormous potential as an
Somatic
alternative method for clonal propagation of large
numbers of rose plants (Rout et al., 1999) and provides
appropriate material for genetic transformation studies
(Verge et al., 2010).
The broad range of regeneration methods that have
been employed with different rose species and cultivars
suggest that it is difficult to develop a general, genotype-
independent protocol for the production of embryogenic
calli and plantlet regeneration (Burrell, 2003). Somatic
embryogenesis has been found to be highly genotype-
dependent in rose (Castillon and Kamo, 2002). There
have been many reports on the formation of somatic
embryos from different explants of rose (Kunitake et al.,
1993; Arene et al., 1993; Matthews et al., 1994; Marchant
et al., 1996; Hsia and Korban, 1996; Kintzios et al., 1999;
Uzunova, 2000; Sarasan et al., 2001; Castillon and Kamo,
2002; Li et al., 2002; Burrell, 2003; Estabrooks et al., 2007;
Zakizadeh et al., 2008; Vergne et al., 2010), but most
protocols apply only to a particular genotype. Despite all
these reports, an efficient system to generate high rates
of somatic embryogenesis remains elusive and
undefined.
Various plant growth regulators (PGRs) have been
used to induce embryogenic calli in different rose species
and cultivars. Different concentrations of auxin alone, or
*Author for correspondence.
combinations of auxins and cytokinins have been used
successfully for somatic embryogenesis in some rose
cultivars (reviewed by Pati et al., 2006).
Transformable explants, having a high rate of
regeneration to whole plants, are the main requirement
for genetic transformation. The frequency of somatic
embryogenesis in rose is generally low, the maximum
reported value being 37% (Zakizadeh et al., 2008). Here,
we have studied the effects of variants PGRs on somatic
embryogenesis in R. hybrida ‘Apollo’ to determine the
best combinations or single PGRs to achieve a high rate
of regeneration for use in our transformation
programme.
MATERIALS AND METHODS
Plant material and culture conditions
Proliferating shoot cultures of R. hybrida ‘Apollo’
were established from nodal stem segments of
greenhouse-grown plants, as described by Hsia and
Korban (1996). All basal media contained half- or full-
strength MS salts (Murashige and Skoog 1962), full-
strength MS vitamins, and 30 g l–1 sucrose, and were
adjusted to pH 5.7 using 1 M NaOH prior to autoclaving
at 103.35 kPa and 121ºC for 15 min. All cultures were
incubated under a 16 h photoperiod provided by cool-
white fluorescent lights (at 60 µmol m–2 s–1), except those
for callus induction which were incubated in the dark,
and at a temperature of 21º ± 1ºC.
Each treatment for callus induction, embryogenic
 400 Somatic embryogenesis in Rosa hybrida L.

callus induction, and embryo maturation was replicated
three times and organised in a completely randomised
design. Each replication consisted of a single 10-cm
diameter Petri plate containing ten leaves or calli.
Embryogenic callus induction
The top four vigorously-growing leaves were excised
from each proliferating shoot. For callus induction,
‘Apollo’ leaves with several cuts across their midribs
were placed on MS medium in three 10-cm Petri dishes,
adaxial-side down. The basal medium, containing full-
strength MS salts and MS vitamins, was supplemented
with 0, 0.5, 1.0, 2.0, or 3.0 mg l–1
-naphthaleneacetic acid
(NAA), alone, or with various combinations of 0, 1.5, or
3.0 mg l–1 zeatin plus 0, 1.0, 2.0, 3.0, 4.0, or 5.0 mg l–1 2,4-
dichlorophenoxyacetic acid (2,4-D), or combinations of
0, 1.0, 2.0, 3.0, 4.0, or 5.0 mg l–1 picloram plus 0, 0.25, or 0.5
mg l–1 kinetin, and were solidified with 2.5 g l–1 Gelrite
(Duchefa Biochemie, Haarlem, The Netherlands).
Cultures were incubated in the dark for 4 weeks. Data on
the number of explants that developed embryogenic calli
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plantlets were then transferred to a 1:1:1 (v/v/v) mix of
soil, peat, and perlite in 4-cm diameter plastic pots for 2
weeks, and covered with a transparent plastic top. The
plastic top was removed gradually, and well-developed
plantlets were transferred to a greenhouse and grown at
23º ± 2ºC. Plants were watered daily using a drip-
irrigation system, and fertilised once every 2 weeks with
250 mg l–1 of a 20–20–20 Crystalon NPK solution
(YARA; Mokhberi Co., Tehran, Iran).
Statistical analysis
All data were analysed using ANOVA. Means were
compared using Duncan’s multiple range test at the 5%
probability level (P ≤ 0.05). All computations were made
using the SAS statistical analysis package (SAS Inc.,
Cary, NC, USA). All percentage data were checked for
–
normal distribution and subjected to arc sine (√ x)
transformation before statistical analysis.
RESULTS AND DISCUSSION

were recorded. Callus started to form 2 weeks after initiating in vitro

leaf explant cultures of R. hybrida ‘Apollo’. Initially, most
Maturation and germination of somatic embryos of the calli formed were compact and white in colour
After 6 weeks in culture, calli produced on the above (Figure 1A), then gradually changed in colour to pale
media were transferred to basal MS medium containing green. The highest percentage (48.3%) of embryogenic
1.0 mg ml–1 ABA alone, or in combination with 0, 2.0, 4.0, callus induction occurred after 4 weeks of culture on full-
or 6.0 mg l–1 6-benzyladenine (BA), or 2.5 or 5.0 mg l–1 strength MS medium containing 1 mg l–1 2,4-D. Media
thidiazuron (TDZ) alone, or in combination with 1.0 mg containing 2.0 or 3.0 mg l–1 2,4-D also showed high rates
l–1ABA. The same medium without any PGR was used as of somatic embryogenic callus production. No
a control. The media were solidified with 2.5 g l–1 Gelrite, embryogenic calli were observed by increasing the
and cultures were incubated for 10 weeks in the light, as concentration of 2,4-D to 4.0 or 5.0 mg l–1. This result
described above. Mature embryos were germinated on agreed with previous reports (Marchant et al., 1996; Kim
full-strength MS medium containing 2.0 mg l–1 BA alone, et al., 2003). All media containing zeatin alone, or zeatin
or 2.0 mg l–1 BA plus 1.0 mg l–1 indole-3-acetic acid plus 2,4-D, resulted in the formation of embryogenic
(IAA). calli. However, the combination of a high concentration
of 2,4-D with zeatin decreased the production of
Plantlet development embryogenic calli (Table I). Vergne et al., (2010) reported
All regenerated shoots derived by somatic using MS medium supplemented with 4.0 mg l–1 zeatin as
embryogenesis were sub-cultured onto PGR-free MS an embryo induction medium. However, media
medium solidified with 3 g l–1 Gelrite for 1 month. The supplemented with higher concentrations of zeatin did
not increase the production of embryogenic calli in
different cultivars of miniature potted rose (Zakizadeh
TABLE I et al., 2008).
Effects of 2,4-D and zeatin on the induction of embryogenic calli from
leaf explants of Rosa hybrida cv. Apollo after 4 weeks in culture
The induction of embryogenic calli occurred on all
media containing NAA, but the percentage of callus
Percentage of leaf
2,4-D Zeatin No. of explants explants forming production decreased with increasing NAA
(mg l–1) (mg l–1) examined embryogenic calli concentration (Table II). Some researchers have
0.0 0.0 30 0.0 f† reported the successful use of different concentrations
0.0 1.5 30 37.7 ± 1.4 bcd of NAA alone, or NAA together with a cytokinin, for
0.0 3.0 30 36.7 ± 4.4 cd
1.0 0.0 30 48.3 ± 1.8 a somatic embryogenesis in Rosa cultivars (Pati et al.,
1.0 1.5 30 31.3 ± 3.7 d 2006). Although, full-strength MS media containing
1.0 3.0 30 30.3 ± 2.4 d
2.0 0.0 30 44.5 ± 3.2 ab
2.0 1.5 30 36.0 ± 2.9 cd TABLE II
2.0 3.0 30 32.0 ± 3.6 d Effect of
-naphthaleneacetic acid (NAA) on the induction of embryo-
3.0 0.0 30 42.0 ± 2.7 abc genic calli from leaf explants of Rosa hybrida cv. Apollo after 4 weeks in
3.0 1.5 30 37.3 ± 1.7 bcd culture
3.0 3.0 30 30.7 ± 2.6 d NAA No. of explants Percent of leaf explants
4.0 0.0 30 0.0 ± 0 f (mg l–1) examined forming embryogenic calli
4.0 1.5 30 16.0 ± 2.5 e
4.0 3.0 30 18.3 ± 1.8 e 0.5 30 45.0 ± 2.4 a†
5.0 0.0 30 0.0 ± 0 f 1.0 30 32.0 ± 1.8 b
5.0 1.5 30 15.0 ± 2.9 e 2.0 30 17.2 ± 1.2 c
5.0 3.0 30 12.3 ± 1.2 e 4.0 30 12.2 ± 0.8 c
† †
Mean values (n = 3) ± SE followed by different lower-case letters Mean values (n = 3) ± SE followed by a different lower-case letter
denote a statistically significant difference at P ≤ 0.05, as determined by denote a statistically significant difference at P ≤ 0.05, as determined by
Duncan’s multiple range test. Duncan’s multiple range test.
 P. AZADI, E. B. ZADEH and V. O. NTUI 401
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FIG. 1
Plant regeneration from somatic embryos derived from in vitro leaf explants of Rosa hybrida cv. Apollo. Panel A, callus induction on full-strength MS
medium containing 1.0 mg l 2,4-D. Panel B, yellowish, friable calli formed on embryo maturation medium containing 1.0 mg l-1 ABA. Panels C, D
–1

cotyledonary bodies induced on yellowish friable calli after 12 weeks. Scale bars = 2.0 mm for Panels A–D. Panel E, plantlet formation after 6 weeks
culture in the light. Panel F, flowering plants after 8 months in a greenhouse. Scale bars = 2.0 cm for Panels E, F.

different combinations of picloram and kinetin produced
embryogenic calli, no embryo maturation was observed
during the next step (data not shown).
Various PGRs have been used for the maturation of
embryogenic calli in different rose cultivars. In the
present study, yellowish friable calli were formed
gradually (Figure 1B) after transferring the calli to
maturation media containing 1.0 mg ml–1 ABA alone, or
in combination with 0, 2.0, 4.0, or 6.0 mg l–1 BA, or
containing 2.5 or 5.0 mg l–1 TDZ alone, or in combination
with 1.0 mg l–1 ABA. The same MS medium without
PGRs was used as the control. A genotypic effect on
callus type has been reported in different miniature rose
cultivars (Zakizadeh et al., 2008). After a further 9 weeks
in culture (i.e., 13 weeks after initiation), cotyledonary
bodies covered most of the yellowish friable calli (Figures
1C, D). The rate of embryo maturation was highest (85.0%)
on medium supplemented with 1.0 mg l–1 ABA alone
(Table III).Applications of ABA alone, or in combination
with gibberellic acid (GA3) have been reported for
embryo maturation (Marchant et al., 1996; Li et al., 2002;
Zakizadeh et al., 2008). Medium containing 2.0 mg l–1 BA
 402 Somatic embryogenesis in Rosa hybrida L.

TABLE III generally higher (73%) on MS medium supplemented

Effect of different concentrations of BA or TDZ alone or in combination
with ABA on the maturation of embryogenic calli of Rosa hybrida cv. with 2.0 mg l–1 BA than on MS medium containing BA
Apollo after 10 weeks in culture plus IAA (57%). Although adding an auxin such as IAA
BA‡ TDZ ABA No. of explants Embryo or indole-3-butyric acid (IBA), and GA3 has been
(mg l–1) (mg l–1) (mg l–1) examined maturation (%) recommended in some reports (Marchant et al., 1996;
0.0 0.0 0.0 20 0.0 c† Zakizadeh et al., 2008), our results showed that
0.0 0.0 1.0 30 85.0 a exogenous BA alone could produce a high rate of
2.0 0.0 0.0 30 50.0 b
2.0 0.0 1.0 30 48.0 b germination in matured embryos (cotyledonary bodies)
4.0 0.0 0.0 30 0.0 c of R. hybrida. After 4 weeks, the embryos elongated and
4.0 0.0 1.0 30 0.0 c
6.0 0.0 0.0 30 0.0 c turned green, but had poorly developed root systems.
6.0 0.0 1.0 30 0.0 c Therefore, they were transferred to bottles containing
0.0 2.5 0.0 20 0.0 c PGR-free MS medium solidified with 3.0 g l–1 Gelrite to
0.0 2.5 1.0 20 0.0 c
0.0 5.0 0.0 20 0.0 c develop better root systems. These cultures were
0.0 5.0 1.0 20 0.0 c maintained in a growth room at 25º ± 1ºC, with a 16 h
†
Mean values (n = 3) ± SE followed by a different lower-case letter photoperiod at 30 – 40 µmol m–2 s–1 from cool-white
denote a statistically significant difference at P ≤ 0.05, as determined by fluorescent lights. Well-developed, rooted plants were
Duncan’s multiple range test.
‡
BA, 6-benzylaminopurine; TDZ, thidiazuron; ABA, abscisic acid. maintained under greenhouse conditions and flowered
after 8 months (Figure 1F). The morphological
characteristics and flower colours of these embryo-
alone, or in combination with 1.0 mg l–1 ABA, also showed derived plants were the same as the donor parent.
embryo maturation (50% and 48%, respectively), while In summary, for optimum embryogenic callus induction
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no maturation was observed by increasing the
concentration of BA. Li et al. (2002) also found low
concentrations of BA or ABA to be more effective for
embryo maturation than high concentrations.
Mature embryos (cotyledonary bodies) were
transferred onto each of two germination media: MS
medium containing 2.0 mg l–1 BA, or MS medium
containing 2.0 mg l–1 BA plus 1.0 mg l–1 IAA. After 6
weeks in culture, in the light, the cotyledonary bodies
formed plantlets on both media (Figure 1E). The
frequency of plantlet production (i.e., the percentage of
cotyledonary bodies which produced plantlets) was
after 4 – 6 weeks in culture, 1.0 mg l–1 2,4-D or 0.5 mg l–1
NAA were the most suitable PGRs in R. hybrida
‘Apollo’. The best medium for embryo maturation was
MS medium containing 1.0 mg l–1 ABA for 9 weeks.
Using MS medium containing 2.0 mg l–1 BA for 4 weeks
gave the highest rate of germination. Transferring
plantlets to PGR-free MS medium solidified with 3.0 g l–1
Gelrite for 4 weeks helped develop the root system,
which was useful before transferring them to soil in a
greenhouse. In total, 21 – 23 weeks were required to
produce whole plantlets in soil. All plants flowered
within 8 months.

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