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Somatic embryogenesis of Pelargonium sidoides DC

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DOI: 10.1007/s11240-015-0726-2

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Plant Cell Tiss Organ Cult
DOI 10.1007/s11240-015-0726-2
ORIGINAL PAPER
Somatic embryogenesis of Pelargonium sidoides DC.
Vijay Kumar • Mack Moyo • Johannes Van Staden
Received: 24 October 2014 / Accepted: 25 January 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract Pelargonium sidoides is a high value medicinal
plant endemic to South Africa and the Lesotho Highlands.
Establishing an efficient regeneration system through so-
matic embryogenesis is necessary for its conservation. An
efficient reproducible protocol for in vitro plant regen-
eration via somatic embryogenesis of Pelargonium si-
doides, an important medicinal plant, is reported for the
first time. Embryogenic callus was obtained on Murashige
and Skoog (MS) basal medium supplemented with
2.0 mg L-1 picloram, 0.5 mg L-1 thidiazuron and
20 mg L-1 glutamine. Different developmental stages of
somatic embryos (SEs: globular embryos, heart shaped
embryos, torpedo shaped embryos and cotyledon embryo
with radicle) were directly obtained and further matured
from embryogenic callus by subsequent subculture on the
same medium. The highest frequency of somatic embryos
(25.89 ± 1.50) was recovered after 6 weeks. Scanning
electron microscopic (SEM) analysis revealed the presence
of embryogenic cell clusters that formed cotyledonary
embryos. Mature somatic embryos germinated and devel-
oped into plantlets after 4 weeks on half-strength MS
medium. High plant regeneration frequency (94.4 %) was
achieved on half-strength MS medium supplemented with
1.0 mg L-1 gibberellic acid. Rooted plantlets were suc-
cessfully acclimatized in the greenhouse with a survival
rate of 90 %. The protocol developed would be helpful in
reducing stress on natural habitats, provide a system for
germplasm conservation, regeneration of large numbers of
high value clonal plants for commercial production, the
V. Kumar
M. Moyo
J. Van Staden (&)
Research Centre for Plant Growth and Development, School of
Life Sciences, University of KwaZulu-Natal Pietermaritzburg,
Private Bag X01, Scottsville 3209, South Africa
e-mail: rcpgd@ukzn.ac.za
isolation of bioactive compounds and genetic transforma-
tional studies.
Keywords Gibberellic acid
Medicinal plant

Pelargonium sidoides
Picloram
Plant regeneration

Somatic embryogenesis
Thidiazuron
Abbreviations
GA3 Gibberellic acid
MS Murashige and Skoog medium
PGRs Plant growth regulators
PPF Photosynthetic photon flux
SEs Somatic embryos
SEM Scanning electron microscopy
TDZ Thidiazuron
Introduction
Pelargonium sidoides DC. (Geraniaceae), a popular med-
icinal plant used in traditional medicine is widely dis-
tributed in South Africa and the Lesotho Highlands. P.
sidoides is extensively used for curing various ailments,
including diarrhoea, colic, gastritis, tuberculosis, cough,
hepatic disorders, menstrual complaints and gonorrhoea
(Brendler and van Wyk 2008; Colling et al. 2010). Two
phytopharmaceutical products, namely, EPsÒ 7630 (Um-
ckaloaboÒ, Dr. Willmar Schwabe GmbH & Co. KG
Pharmaceuticals, Germany) and LinctagonÒ (Nativa, South
Africa) are produced from P. sidoides root extracts. High
demand of this plant species in both national and interna-
tional markets presents an opportunity for researchers to
investigate novel approaches for its in vitro cultivation and
conservation. According to the Red List of South African
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 Plant Cell Tiss Organ Cult

Table 1 The effect of Growth regulators in solid MS medium (mg L-1) Somatic embryogenesis (%) Mean number of SEs
Picloram, Thidiazuron and
Glutamine on embryogenic Picloram TDZ Glutamine
callus induction and somatic
embryo development in P. Control 0.0 0.0 0 0
sidoides 0.5 0.5 0.0 77.96 ± 3.1f 9.11 ± 0.56e
1.0 0.5 0.0 80.44 ± 3.5e 13.78 ± 0.40cd
2.0 0.5 0.0 93.89 ± 2.6c 18.22 ± 1.19b
3.0 0.5 0.0 96.33 ± 1.8b 14.33 ± 0.52cd
4.0 0.5 0.0 90.22 ± 2.8d 12.22 ± 0.90cd
5.0 0.5 0.0 81.67 ± 2.5e 11.67 ± 0.55d
0.5 0.5 20 100 ± 0.00a 12.22 ± 0.57cd
1.0 0.5 20 100 ± 0.00a 18.44 ± 1.06b
2.0 0.5 20 100 ± 0.00a 25.89 ± 1.50a
Means followed by same letters
in each column are not 3.0 0.5 20 100 ± 0.00a 20.00 ± 0.88b
significantly different according 4.0 0.5 20 100 ± 0.00a 14.56 ± 0.70c
to Duncan’s multiple range test 5.0 0.5 20 91.44 ± 1.6d 13.33 ± 1.21cd
(P B 0.05)

Fig. 1 Embryogenic callus formation and developmental stages of embryos. c Heart-shaped embryo. d Induction of cotyledonary
somatic embryos of P. sidoides. a Proliferation of embryogenic callus embryo. e Cotyledonadry-shaped embryo. f Cotyledonary somatic
on MS medium containing 2.0 mg L-1 Picloram and 0.5 mg L-1 embryo showing shoot apex and leaf primordial. Scale Bar
TDZ and 20 mg L-1 Glutamine. b Globular stages of somatic a = 10 mm; b–e = 2 mm; f = 5 mm

plants version 2013, P. sidoides conservation status has
been characterised as ‘least concern’ (http://redlist.sanbi.
org/).
Although micropropagation and conservation strategies
for P. sidoides have been described (Moyo et al. 2012,
2013) there is no documentation on somatic embryogenesis.
Somatic embryogenesis is one of the most promising ap-
proaches for plant propagation due to production of large
numbers of plantlets (Martin 2004), the possibility of
producing synthetic seeds (Manjkhola et al. 2005; Kumar
and Chandra 2014), the ability to store and rapidly mobilize
germplasm for cryopreservation (Hua and Rong 2010), the
opportunity for genetic manipulation (Pathi et al. 2013) and
production of bioactive compounds within a short period of
time (Aderkas et al. 2002; Jeong et al. 2005). It is necessary
to develop a method for mass clonal propagation and con-
servation to satisfy the pharmaceutical demand of this high
value medicinal plant. The present investigation was

123
Plant Cell Tiss Organ Cult
Fig. 2 Somatic embryos of P. sidoides at different stages of
development (viewed under stereo and scanning electron microscope)
a Globular-shaped somatic embryo. b Heart-shaped somatic embryo.
c Early torpedo- shaped embryo. d Torpedo stage of somatic embryo.
e Early cotyledonary stage of embryo. f Cotlyledonary embryo with
undertaken with the objective of developing an efficient
in vitro somatic embryogenesis protocol for P. sidoides.
Materials and methods
Plant material and culture initiation
BenlateÒ and agar bacteriological powder were purchased
from Du Pont de Nemours Int., South Africa and Oxoid
Ltd., Basingstoke, Hampshire, England, respectively. Pi-
cloram, Thidiazuron (TDZ), Glutamine, myo-inositol, vi-
tamins (thiamine HCl, nicotinic acid, pyridoxine HCl) and
glycine, were obtained from Sigma–Aldrich Co., Stein-
heim, Germany. All chemicals used in the assays were of
analytical grade.
Mature P. sidoides DC. plants were collected from
University of KwaZulu-Natal (UKZN) Botanical Gardens,
Pietermaritzburg, South Africa. For culture initiation leaf
explants were rinsed thoroughly under running tap water,
and surface decontaminated in 70 % alcohol (v/v) for
1 min followed by 2 % sodium hypochlorite (v/v) with
radicle. g Scanning electron micrograph showing the appearance of
embryogenic callus. h Globular somatic embryos formed from
embryogenic callus. i Cluster of globular shaped somatic embryos.
j Torpedo-shaped somatic embryo. k Cotyledonary-shaped somatic
embryo. Scale Bar a–f = 2 mm; g, k = 1 mm; h–j = 200 lm
Tween 20 for 20 min. The explants were thoroughly rinsed
three times with sterile distilled water, excised into 10 mm
sections and inoculated on MS (Murashige and Skoog
1962) medium supplemented with 30 g L-1 sucrose,
0.1 g L-1 myo-inositol and different concentrations and
combinations of plant growth regulators [PGRs: picloram
(0.5–5 mg L-1), and thidiazuron (0.5 mg L-1) and
20 mg L-1 glutamine for production of somatic embryos
(SEs). The pH of the medium was adjusted to 5.8 with
0.1 N KOH and/or 0.1 N HCl before solidification with
8 g L-1 agar. Aseptic cultures were maintained at
25 ± 2 °C, 16-h photoperiod and photosynthetic photon
flux (PPF) of 40 lmol m-2 s-1 provided by cool white
fluorescent tubes (OSRAM L 58 W/640, Germany). In all
experiments, the medium without plant growth regulators
served as control.
Embryogenic callus induction
Calli obtained from P. sidoides cultures were inoculated on
MS media with different concentrations and combinations
of picloram with TDZ and glutamine, as specified in
123

Fig. 3 Germination from somatic embryos on germination medium. a Cotyledon somatic embryos. b Germinated embryo with radicle. c, d
Germination of somatic embryos. Scale Bar a–b = 1 cm; c–d = 2 cm
Table 1 for embryogenic callus induction. The media were
solidified with 8 g L-1 agar powder and 30 g L-1 sucrose
was added as a carbon source.
Friable, embryogenic calli were selected and transferred
to MS medium supplemented with 2.0 mg L-1 picloram,
0.5 mg L-1 TDZ and 20 mg L-1 glutamine. Different
developmental stages of somatic embryos (SEs: globular,
heart, torpedo and cotyledonary) were recorded after
6 weeks of culture. All embryo stages were photographed
using a Leica M Stereo Microscope (JVC-Digital Camera:
KY-F 1030U type; 0.5X, Wayne, NJ, USA) (Fig. 2a–f).
Scanning electron microscopy
Scanning electron microscopy (SEM) was used for mor-
phological observation of SEs. For SEM analysis, SEs were
fixed in 2.5 % glutaraldehyde for 1 h and dehydrated
through a graded ethanol series (10, 30, 50, 70, 90 and
100 %) sequentially for 10 min each with two changes.
Dehydrated tissues was then dried in a critical point dryer
(Quorum K850) and coated with gold in an ion coater
(Eiko IB-3 ion coater, Japan). The samples were examined
under a scanning electron microscope ZEISS EVOÒ MA
15 SEM (Carl Zeiss SMT, Germany) and photographs were
taken at different magnifications.
123
Plant Cell Tiss Organ Cult
Germination of somatic embryos and plantlet
regeneration
Modifications were made in MS medium by reducing the
salt concentrations, sucrose level and addition of amino
acids. To regenerate whole plants, cotyledonary somatic
embryos were transferred to plant regeneration medium
[half-strength solid MS medium and full strength solid MS
medium plus 1.0 mg L-1 gibberellic acid (GA3)]. For so-
matic embryo conversion and plant growth, the cultures
were maintained at a PPF of 40 lmol m-2 s-1 provided by
cool white lamps with a 16 h photoperiod. Embryo ger-
mination percentage was recorded after 4 weeks.
Ex vitro acclimatization
Twenty in vitro grown plantlets with well-developed roots
were removed after 4 weeks, washed thoroughly with
running tap water to remove culture medium residue and
planted in plastic pots containing a 1:1 (v/v) vermiculite:
sand mixture and placed in an environmentally controlled
misthouse (90–100 % relative humidity; average mid-day
PPF, 90–100 lmol m-2 s-1) under natural photoperiod
conditions. After 2 weeks, the acclimatized plants were
successfully transferred and established in a greenhouse
Plant Cell Tiss Organ Cult
Fig. 4 Plantlet formation from somatic embryos on germination medium. a, b Plant showing healthy shoots and roots just before transfer to soil.
c, d Acclimatized plants of P. sidoides in the green house. Scale Bar a = 2 cm
under natural photoperiod conditions, temperature
(25 ± 2 °C) and mid-day PPF of approximately
1,000 lmol m-2 s-1.
Statistical analysis
In this study, each treatment was repeated in three repli-
cated experiments with 20 explants per treatment. The data
are presented as mean ± standard error (Mean ± SE). All
data were analyzed using ANOVA, and means were
separated using the Duncan’s multiple range test (DMRT)
at P B 0.05 (IBM SPSS ver. 16).
Results and discussion
In the present study, an efficient and highly reproducible
system for P. sidoides somatic embryogenesis was devel-
oped (Fig. 1a–f). Somatic embryogenesis was achieved from
leaf explants on MS medium, 3 % (w/v) sucrose and PGRs:
picloram (0.5–5 mg L-1), and thidiazuron (0.5 mg L-1)
and 20 mg L-1 glutamine (Table 1). Embryogenic calli
formed on leaf explants after a week in culture and later
globular embryoids developed directly from leaf explants in
all treatments except the control after 4 weeks (Fig. 1). The
addition of TDZ with picloram significantly increased the
formation of somatic embryos. Furthermore, combination of
picloram, TDZ and glutamine induced high production of
SEs (Table 1). The addition of an organic nitrogen form has a
positive influence on somatic embryogenesis (Robichaud
et al. 2004; Zouine and Hadrami 2007; Gerdakaneh et al.
2011). Glutamine has been shown to improve somatic em-
bryogenesis in both monocotyledons and dicotyledons (Dhir
et al. 1991; Yin et al. 1993; Ke et al. 1996; Vani and Reddy
1996; Shrivastava and Chawla 2001). The addition of glu-
tamine (20 mg L-1) in media influenced effective SEs for-
mation in P. sidoides (Table 1; Fig. 1). Similarly, several
reports confirmed that formation of SEs was enhanced by
glutamine (Baskaran and Jayabalan 2009; Deo et al. 2010;
Gerdakaneh et al. 2011). The role of picloram, TDZ and
123
 Plant Cell Tiss Organ Cult

strength MS medium (Fig. 5). Similar results have been

reported in other plant species (Paul et al. 2011; Raju et al.
100 a
2013; Khan et al. 2015). The addition of 1.0 mg L-1 GA3
Embryo Germination (%)

b to the medium significantly increased the germination of

80
c embryos. The beneficial effect of GA3 on SE germination
has been reported for other plant species (Cangahuala
60
d Inocente et al. 2007; Sivanesan et al. 2011; Siddiqui et al.
2011; Peńa-Ramı́rez et al. 2011; Raomai et al. 2014).
40
The plantlets were successfully acclimatized in a
20
greenhouse under ex vitro conditions (Fig. 4c–d). The ac-
climatization procedure resulted in a survival rate of 90 %.
0
The hardened plants were found to be healthy. There was
S S A
3
A
3 no observable phenotypic variation between the original
M M l G l G
th th g/ g/ greenhouse-grown plants and those regenerated from so-
ng ng 1m 1m
tre tre + + matic embryos in this study, showing that the regenerated
fS l lS S S
al Fu
M M
H th th plantlets were true-to-type.
ng ng
tre tre
fS l lS
al Fu
H

Fig. 5 The effect of half-strength MS, full-strength MS and GA3 on Conclusions

the germination of somatic embryos of P. sidoides. The data were
recorded after 4 weeks of culture. Mean ± SE followed by the same We have successfully established an efficient plant regen-
letter are not significantly different at the 0.05 % level as determined eration system via somatic embryogenesis from leaf ex-
by Duncan’s multiple range test
plants of P. sidoides. This is the first report that defines
successful somatic embryogenesis in P. sidoides and also
glutamine on SEs production has also been reported in other suggests a novel protocol for the conditions necessary to
plant species (Tribulato et al. 1997; Baskaran and Van Sta- induce embryogenesis and in vitro regeneration in this
den 2012, 2014). plant. Using this method, large number of plants can be
SEM analysis of embryogenic callus obtained from MS easily propagated which could greatly speed up the process
medium supplemented with 2.0 mg L-1 picloram, 0.5 mg L-1 of mass clonal propagation for conservation of this valu-
TDZ and 20 mg L-1 glutamine showed the development of able medicinal plant and as a potential source for produc-
somatic embryos in an asynchronous way. In other words, dif- tion of bioactive compounds.
ferent developmental stages of embryos were obtained on the
same cultured explant. The use of SEM revealed the presence of Acknowledgments V. Kumar and M. Moyo thank the National
globular embryos on the surface of explants (Fig. 2g–i). It also Research Foundation and the University of KwaZulu-Natal, South
Africa for support in the form of postdoctoral fellowships. The au-
showed the torpedo stage somatic embryo with a distinct shoot thors are grateful to the Microscopy and Microanalysis Unit (MMU),
tip and glandular hairs throughout its surface (Fig. 2j) and the UKZN, Pietermaritzburg for microscopic assistance.
cotyledonary stage characterized by well-differentiated cotyle-
dons with a radicle (Fig. 2k).
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