Plant Cell, Tissue and Organ Culture (2005) 80: 33–42
Springer 2005

Development of a shoot regeneration protocol for genetic transformation in

Pelargonium zonale and Pelargonium peltatum hybrids

Traud Winkelmann*, Kamran Kaviani & Margrethe Serek

Institute of Floriculture, Tree Nursery Science and Plant Breeding, Section of Floriculture, University of
Hannover, Herrenhaeuser Strasse 2, D-30419 Hannover, Germany (*requests for offprints; Fax: +49-511-
762 2654; E-mail: traud.winkelmann@zier.uni-hannover.de)

Received 23 February 2004; accepted in revised form 6 April 2004

Key words: Agrobacterium-mediated gene transfer, breeding, geranium, in vitro culture,

Pelargonium · hortorum, regeneration ability

Abstract

The attempts of this investigation were to develop a system for plant regeneration from explants of adult
plants and its use for genetic transformation of important commercial Pelargonium zonale hybrid and P.
peltatum hybrid cultivars. To this aim, leaf blade and petiole explants of eight cultivars were cultured on
modified MS (Murashige and Skoog, 1962) medium with two concentrations of TDZ, BA, and zeatin (5
and 20 lM). Petiole explants showed a higher regeneration response than leaf blade explants and TDZ was
the most effective cytokinin. Petioles of 16 cultivars were incubated on medium containing 5, 10, 15, and
20 lM TDZ, respectively, in order to identify the most effective TDZ concentration. For the majority of
genotypes 10 lM TDZ resulted in the best regeneration response. Cefotaxim at 500 mg l)1 had no effect on
shoot regeneration and did not show interaction with glufosinate. For selection of transgenic cells, a
concentration of 2.5 lM glufosinate was shown to be appropriate. LBA4404 and EHA101 Agrobacterium
strains carrying pIBGUS vector with pat gene as selectable marker gene and GUS gene as reporter gene
were compared in transformation studies. With regard to GUS expression in petiole explants 16 days after
coculture, better results were obtained with EHA 101 than with LBA 4404.

Abbreviations: BA – 6-benzylaminopurine; GUS – b-glucuronidase; TDZ (=thidiazuron) – N-1,2,3-(thi-

diazol-5-yl)urea; zeatin – 6-(4-hydroxy-3-methyl-2-butenylamino)purine

Introduction marketing quality, and pest and disease resistance,

Pelargonium is a genus within the Geraniaceae out
of which some species and interspecific hybrids are
horticulturally used as potted or bedding plants of
considerable economic importance. In the Euro-
pean and North American markets the vegeta-
tively-propagated bedding plants from the two
groups Pelargonium zonale hybrids (syn. Pelargo-
nium · hortorum) and Pelargonium peltatum hy-
brids are predominant. Breeding aims at
developing new flower colours and shapes, early
and continuous flowering, good postharvest and
among others. Although breeding resulted in a
range of cultivars with excellent traits, some of
these aims have not yet been reached, and others,
such as resistance against viral or bacterial diseases
cannot be achieved by conventional cross-breeding
strategies. Gene transfer using Agrobacterium
tumefaciens enables the introduction of genes from
outside the related species and would be a helpful
tool in pelargonium breeding. A prerequisite for
applying genetic transformation via Agrobacte-
rium is an in vitro regeneration system. For prac-
tical application, it is strongly recommended that
34

explants can be obtained from adult plants, be-
cause the genotypes have to be tested for agro-
nomic performance before using them for
transformation, and, moreover the vegetatively-
propagated pelargoniums are highly heterozygous
resulting in variable offspring. For P. zonale as
well as P. peltatum hybrids, a number of reports
has been published which dealt with in vitro
regeneration from explants taken from adult
plants either using organogenesis (Cassells and
Carney, 1987; Dunbar and Stephens, 1989; Boase
et al., 1996; Robichon et al., 1997; Agarwal and
Ranu, 2000; Mithila et al., 2001) or using somatic
embryogenesis (Cassells, 1979; Robichon et al.,
1997; Haensch, 1999; Mithila et al., 2001). In the
case of somatic embryogenesis, the most restricting
problem is the applicability of the system, which
has been shown to work efficiently for a few model
genotypes (KrishnaRaj et al., 1997), but was not
transferable to a wider range of genotypes of ac-
tual economic importance (Haensch, 1999). In
addition, recently it was questioned whether some
of the reports on somatic embryogenesis were
concerned with organogenesis rather than
embryogenesis (Haensch, 2004). However, the re-
ports on shoot regeneration in pelargonium also
stress the differences between genotypes (Robi-
chon et al., 1997; Mithila et al., 2001). Agrobac-
terium-mediated transformation of pelargonium
types which are in focus of this study was only de-
scribed by Boase et al. (1996, 1998). Besides, few
other reports are available dealing with gene
transfer to other pelargonium types, such as scented
pelargoniums (Pellegrineschi and Davolio-Mariani,
1996; KrishnaRaj et al., 1997; Bi et al., 1999).
Objectives of this study were to develop and test an
in vitro regeneration system for a spectrum of dif-
ferent cultivars of P. zonale and P. peltatum hybrids
using explants from adult plants. Additionally, the
regeneration system was used in transformation
experiments with two Agrobacterium tumefaciens
strains testing different co-culture durations.
Material and methods
Plant material
The 16 pelargonium cultivars collected from three
German breeders used in this study are listed in
Table 1. Nine cultivars belong to P. zonale hy-
brids, seven to P. peltatum hybrids. They were
grown in the greenhouse at 18–22 C and explants
were taken from healthy non-flowering plants.
Surface sterilisation and growth conditions
Leaves and petioles were surface sterilised in
500 ml sodium hypochlorite solution with 1.5%

Table 1. Pelargonium cultivars used: some characteristics and breeders

Internal No. Cultivar name Pelargonium group Flower colour Breeder

P01 Sailing P. zonale hybrid White Selecta Klemm

P02 Caprivi P. zonale hybrid White Selecta Klemm
P03 Salmon Flash P. zonale hybrid Pink Selecta Klemm
P04 Katinka P. zonale hybrid Pink Selecta Klemm
P05 Arcona 2000 P. zonale hybrid Red Selecta Klemm
P06 Royal Pink P. peltatum hybrid Red Selecta Klemm
P07 Royal Blue P. peltatum hybrid Red Selecta Klemm
P08 Ville de Paris P. peltatum hybrid Red Selecta Klemm
P09 PAC Granatit, Penro P. peltatum hybrid Red Elsner PAC
P10 PAC Dresdner Rhodonit,P. peltatum hybrid Red Elsner PAC
Paccherry
P11 PAC Lauretta, Pacamla P. zonale hybrid Pink Elsner PAC
P12 PAC Juliane, Penjul P. zonale hybrid Red Elsner PAC
P13 Pelfi Rokoko P. zonale hybrid Pink Pelargonien Fischer
P14 Pelfi Grand Prix P. zonale hybrid Red Pelargonien Fischer
P15 Pelfi Beach 99 P. peltatum hybrid Red Pelargonien Fischer
P16 Pelfi Amethyst 96 P. peltatum hybrid Red Pelargonien Fischer

available chlorine containing one drop of Tween
20 for 10 min, followed by three washing steps in
sterile water for 1, 2 and 5 min. Petioles were cut
into segments of 4–6 mm length and leaf blade
explants were 5 · 5 mm in size. While petiole
segments were placed horizontally on the medium,
leaf explants were incubated with the adaxial side
facing up in 6 cm plastic Petri dishes containing
10 ml medium. The Petri dishes were sealed with
Parafilm and all cultures were kept at 23–25 C
under 16 h of light (20–40 lmol m)2 s)1).
Culture media
The basal medium composition contained micro-
nutrients according Murashige and Skoog (1962),
36.71 mg l)1 NaFeEDTA, 250 mg l)1 peptone,
100 mg l)1 myo-inositol, 2 mg l)1 glycine,
0.5 mg l)1 nicotinic acid, 0.5 mg l)1 pyridoxine
HCl, 0.1 mg l)1 thiamine HCl, 30 g l)1 sucrose
and 8 g l)1 Agar (Serva Nr. 11396). The pH was
adjusted to 5.8 prior to autoclaving. According to
Reuther (1983) different macronutrients were used
for P. zonale and P. peltatum hybrids: For P. zo-
nale hybrids, macronutrients of full-strength MS
medium were supplied, but with 2470 mg l)1
KNO3 and 255 mg l)1 KH2PO4. Media for P.
peltatum hybrids contained MS macronutrients at
half-strength, but with 1235 mg l)1 KNO3 and
127.5 mg l)1 KH2PO4. Cytokinin concentrations
are given below; auxin content of all media was
kept constant at 2.85 lM IAA for P. zonale hy-
brids and 0.57 lM IAA for P. peltatum hybrids.
Regeneration experiments
For each treatment in all experiments, five Petri
dishes with six explants each were prepared. All
experiments were repeated twice. After four weeks,
the number of explants with shoots and the number
of shoots per explant were counted. Shoot regen-
eration is given in percentage of explants
with shoots based on the total number of sterile
explants.
Effects of cytokinin type and explant type on shoot
regeneration
The three cytokinins BA, zeatin and TDZ (which
is not a cytokinin in a narrow sense, but provokes
35
cytokinin-like effects and in this study will be
nominated as cytokinin) were applied at 5 and
20 lM, respectively. As explants, petiole segments
and pieces of the leaf blade were compared for
pelargonium cultivars P01, P04, P06, P08, P10,
P11, P14, and P15 (Table 1).
Effect of TDZ concentration on shoot regeneration
from petiole explants
To limit the scope for the optimal TDZ concen-
tration and to screen all sixteen cultivars, this
experiment was performed using petiole segments
as explants. TDZ was tested in concentrations of 5,
10, 15, and 20 lM, respectively.
Effect of glufosinate and cefotaxim on shoot
regeneration
In order to identify appropriate concentrations of
glufosinate (synonym phosphinothricin) as selec-
tive agent and to check the interaction with cefo-
taxim used for suppression of agrobacteria after
co-culture, petiole explants of the four pelargo-
nium cultivars P01, P04, P07, and P09 were incu-
bated on basal media for P. zonale and P. peltatum
hybrids with 10 lM TDZ. Because peptone could
interfere with the selection procedure, its effect on
shoot regeneration was tested. To media without
peptone, glufosinate was added at 2.5, 5, 10 and
25 lM as filter-sterilized stock solution after
autoclaving. In a further experiment, the addi-
tional effect of 500 mg l)1 cefotaxim in media with
2.5 and 5 lM glufosinate was investigated.
Transformation experiments
Two Agrobacterium strains EHA101 and
LBA4404 carrying the disarmed plasmid pIBGUS
were used for transformation experiments. This
plasmid is derived from the binary vector pBIN19
(Bevan, 1984) and it harbours between its right
and left borders the T-DNA carrying the nptII
(neomycin phosphotransferase II) gene under nos
(nopaline synthase) promoter, the pat (phosphi-
nothricin acetyl transferase) gene under 35S pro-
moter and the GUS INT (b-glucuronidase with
intron) gene under 35S promoter.
Twenty millilitres of LB medium (Maniatis
et al., 1982) containing 50 mg l)1 kanamycin were
inoculated with 30 ll of frozen glycerine cultures
of Agrobacterium and incubated at 28 C in
36

darkness on a shaker at 250 rpm. After 24 h
optical density was measured at 600 nm and bac-
teria were pelleted at 3000 rpm for 10 min, the
supernatant was discarded and replaced by liquid
hormone-free plant culture medium (1/2 MS) and
Acetosyringone (0.05 mM) in the manner of
adjusting OD600 to 0.2. The solution obtained was
used for explant inoculation.
Petiole explants of cultivars P01, P04, P07, and
P09 were incubated for 10 min in this solution,
then blotted dry on sterile filter paper and placed
on basal medium containing 10 lM TDZ for co-
culture. After 2 and 4 days of co-culture they were
transferred to medium of the same composition
with additional 500 mg l)1 cefotaxim and 2.5 lM
glufosinate.
Histochemical GUS assay according to Jeffer-
son (1987) was done after 16 days with 12–19 ex-
plants per treatment and the number of blue spots
was evaluated.
Statistical analyses
Since the data showed neither normal distribution,
nor homogeneity of variances, they were analysed
by SAS software using nonparametric Brunner
procedure and Kruscal–Wallis test (Brunner,
2001).
Results
The regeneration experiments were aimed at
identifying conditions for shoot regeneration in a
wide spectrum of genotypes. The surface sterilisa-
tion procedure was effective, as was concluded
from the low percentage of contaminated explants
in all experiments (1.7–7.5%).
Regeneration experiments
Effects of cytokinin type and explant type on shoot
regeneration
In the comparison of three different cytokinins
regarding their efficiency in inducing shoot regen-
eration, BA was least promotive. On BA-con-
taining media, only two of the eight cultivars
regenerated shoots (Table 2). The most potent
cytokinin was TDZ leading to shoot regeneration
in seven out of the eight cultivars. Of the two TDZ
concentrations tested (5 and 20 lM), neither was
identified as superior for the majority of genotypes
and therefore an additional experiment was per-
formed to find out the most appropriate concen-
tration. Not only the number of explants with
shoot regeneration, but also the number of shoots
per explant was greatest for TDZ-containing
media (data not shown). Zeatin induced shoot
formation in half of the genotypes tested and the
shoots were of good quality. However, also the
shoots from TDZ media were rooted on hormone-
free medium.
Eight cultivars were included in this experi-
ment; in seven of them shoot regeneration was
obtained (Table 2), only for the P. zonale cultivar
P14 no shoots were induced, irrespective of the
cytokinin. In general, cultivars of the P. peltatum
group expressed greater shoot regeneration ability

Table 2. Shoot regeneration rates [%] of seven Pelargonium cultivars (best medium for each genotype and kind of explant in bold)

Medium

5 lM BA 20 lM BA 5 lM TDZ 20 lM TDZ 5 lM zeatin 20 lM zeatin

Petiole Leaf Petiole Leaf Petiole Leaf Petiole Leaf Petiole Leaf Petiole Leaf

P. zonale hybrids
P01 0 0 0 0 0 0 7 0 0 0 0 0
P04 0 0 0 0 47 60 45 13 23 27 7 27
P11 0 0 0 0 0 0 13 17 0 0 0 0

P. peltatum hybrids
P06 0 0 0 0 47 0 17 20 20 0 10 17
P08 0 0 0 0 50 17 37 23 17 0 46 0
P10 20 0 0 0 0 13 0 0 0 0 0 0
P16 33 3 10 0 23 20 0 100 33 0 17 0

than P. zonale hybrids. For the majority of geno-
types, petiole explants were superior compared to
leaf blade explants, in shoot formation (Table 2).
Therefore, for the subsequent experiments petiole
explants were used.
Effect of TDZ concentration on shoot regeneration
from petiole explants
All 16 genotypes were involved in testing the
concentrations of 5, 10, 15, and 20 lM TDZ for
shoot formation and significant differences were
found between the cultivars as well as between
TDZ concentrations. Regarding the shoot regen-
eration rate for some genotypes (P01, P04, P09)
there was no difference between 5 and 10 lM TDZ
(Figure 1), but a clear decrease at the higher con-
centrations. For these genotypes the optimal TDZ
concentration is expected to lie between 5 and
10 lM. On the other hand, for two genotypes with
low regeneration rates, 10 lM TDZ was necessary
for shoot formation (cultivar P07 presented in
Figure 1 and P16). In addition, the number of
shoots being formed per explant was higher in
some genotypes, if the medium contained 10 lM
TDZ as compared to 5 lM. Therefore, 10 lM
TDZ were applied in the subsequent experiments.
The reaction of all genotypes on medium with
10 lM TDZ is presented in Figure 2. In eight of
the sixteen cultivars, shoot regeneration was ob-
served. If P. peltatum and P. zonale cultivars are
60
50
Shoot regeneration [%]
40
30
20
10
0
5 10 15 20 5 10 15 20
µM µM µM µM µM µM µM µM
P01 P04
P. zonale hybrids
Figure 1. Effect of TDZ on shoot regeneration of four pelargonium cultivars (TDZ concentrations are given in lM).
37
opposed, in the latter group a smaller number of
reacting cultivars (three of nine) was found, but
with higher percentages of regenerating explants
(30–57%) and with higher numbers of shoots per
explant (Figure 2). On the other hand, in the case
of P. peltatum hybrids, more regenerative cultivars
were observed (five of seven). In an additional
experiment, explants taken from in vitro grown
shoots, when tested under the same conditions,
showed higher shoot regeneration rates of up to
83% (results not shown).
Effect of glufosinate and cefotaxim on shoot
regeneration
With the aim of identifying a concentration of
glufosinate appropriate for the later selection of
transgenic shoots, petiole segments of four culti-
vars were incubated on six different media, with or
without peptone and with increasing concentra-
tions of 2.5, 5, 10 and 25 lM glufosinate. The
comparison of the two media lacking the selective
agent revealed that peptone neither improved
callus formation nor shoot regeneration (Fig-
ure 3), and could therefore be omitted from the
medium. Glufosinate was a potent selective agent
being efficient in the lowest concentration
(2.5 lM), at which only few explants formed callus
and no shoots were observed (Figure 3). At the
higher concentrations no callus or shoot formation
occurred and the explants became necrotic within
5 10 15 20 5 10 15 20
µM µM µM µM µM µM µM µM
P07 P09
P. peltatum hybrids
38

80
6.9
data above columns = mean standard deviation
number of shoots per shoot
70 forming explant

60
shoot regeneration [%]

50

1.2
40
1.9
1.6
30
2.0

20
1.5 1.5 1.5

10

0 0 0 0 0 0 0 0
0
P01 P02 P03 P04 P05 P11 P12 P13 P14 P06 P07 P08 P09 P10 P15 P16

P. zonale hybrids P. peltatum hybrids

Figure 2. Shoot regeneration of 16 Pelargonium cultivars after four weeks culture on medium containing 10 lM TDZ (means and
standard deviations of five Petri dishes).

three to four weeks. The statistical analysis re- both agrobacterium strains. The results of the
vealed significant differences between the media GUS assays for the four cultivars 16 days after co-
(p > 0.99), but the pair-wise comparison of media culture are given in Table 4. For strain LBA 4404
with or without peptone showed no significant the prolonged co-culture of 4 days revealed higher
differences regarding callus formation or shoot numbers of 26 explants showing GUS activity
regeneration. summed for the four cultivars as compared to 14
The addition of cefotaxim had no effect on callus for the 2-days co-culture variant. In addition, for
formation, while the shoot regeneration rate slightly each genotype the average number of blue spots
increased in cultivars P04 and P09 and decreased in per explant was increased by 4 days co-culture as
P01 and P07 (Table 3). In total, this effect of cefo- compared to 2 days (Table 4). For the more vir-
taxim was not statistically significant. The combi- ulent strain EHA 101, the four days co-culture
nation of 500 mg l)1 cefotaxim and 2.5 lM period led to very strong bacterial growth, which
glufosinate resulted in markedly decreased callus could not be suppressed by 500 mg l)1 cefotaxim,
formation and completely inhibited shoot formation so that all explants in this treatment were lost.
(Table 3) in the same way that was observed for After 2 days of co-culture with strain EHA 101, 31
glufosinate alone (Figure 3). At 5 lM glufosinate out of 44 explants were found to express GUS
and 500 mg l)1 cefotaxim neither callus nor shoot activity giving a percentage of 70%, which was
formation was observed (Table 3). From these re- about three times greater than for strain LBA 4404
sults it can be concluded that cefotaxim did not showing blue spots in 14 out of 60 explants (Ta-
interfere with the selective effect of glufosinate. For ble 4).
the subsequent transformation experiments, after In a second experiment, the higher virulence
co-culture, petiole explants were incubated on estimated by the GUS expression within explants
medium lacking peptone and containing 10 lM of strain EHA 101, compared to LBA 4404, was
TDZ, 2.5 lM glufosinate and 500 mg l)1 cefotaxim. confirmed (data not shown). However, in this
experiment the co-culture time was 2 versus
Transformation experiments 3 days, respectively. The longer co-culture time of
3 days had little positive effect on the number of
In the first transformation experiment, two co- explants with GUS activity, but increased the
culture times of 2 and 4 days were compared using average number of transformation events per ex-
 39

100

90
P01
P04
80
P07

70 P09
Callus formation [%]

60

50

40

30

20

10

0
C1 C2 AB C D
(a) Medium

40

35 P01
P04

30 P07
P09
Shoot regeneration [%]

25

20

15

10

0
C1 C2 AB C D
(b) Medium

Figure 3. Effect of peptone and glufosinate on callus formation (a) and shoot regeneration (b) of petiole explants of four Pelargonium
cultivars after four weeks, C1: with 250 mg l)1 peptone, C2: without peptone, A: C2 with 2.5 lM glufosinate, B: C2 with 5 lM
glufosinate, C: C2 with 10 lM glufosinate, D: C2 with 25 lM glufosinate (means and standard deviations of five Petri dishes).

plant, as evaluated by counting the number of blue types are the prerequisite for developing a trans-
spots per petiole. formation system for a plant species or genotype. In
pelargonium, many reports on shoot regeneration
and somatic embryogenesis are available, which
Discussion used seedlings or zygotic embryos as starting
material (e.g. Marsolais et al., 1991; Gill et al., 1994;
Regeneration experiments Hutchinson et al., 1996; Murthy et al., 1996; Wilson
et al., 1996; Croke and Cassells, 1997) and which
Efficient regeneration protocols that could be have been successfully applied in transformation
applicable to a broad spectrum of different geno- studies. But the two groups included in this study,
40

Table 3. Effect of cefotaxim (cef) alone and combined with glufosinate (glu) on callus formation and shoot regeneration of four
Pelargonium cultivars

Medium Parameter Cultivar

P01 P04 P07 P09

Control Callus formation [%] 100 100 100 100

Shoot regeneration [%] 20 27 20 10

500 mg l)1 cef Callus formation [%] 100 100 100 100
Shoot regeneration [%] 7 30 17 13

500 mg l)1 Callus formation [%] 13 3 17 13

cef + 2.5 lM glu
Shoot regeneration [%] 0 0 0 0

500 mg l)1 Callus formation [%] 0 0 0 0

cef + 5.0 lM glu
Shoot regeneration [%] 0 0 0 0

Table 4. Histochemical GUS assay in petiole explants of Pelargonium cultivars 16 days after co-culture with two Agrobacterium strains

Strain Cultivar Co-culture Number of Number of explants Average number

period (days) explants analysed with blue spots of blue spots
per explant

LBA 4404 P01 2 17 9 2

4 17 6 2.17
P04 2 12 4 1.75
4 12 2 7
P07 2 13 1 1
4 19 10 1.1
P09 2 18 0 0
4 18 8 2
EHA 101 P01 2 18 12 1.58
a a a
4
P04 2 2 1 16
a a a
4
P07 2 6 5 1.6
a a a
4
P09 2 18 13 2.15
a a a
4
a
Explants overgrown by Agrobacterium, not assayed.

P. zonale and P. peltatum hybrids, are propagated
vegetatively via cuttings and are highly heterozy-
gous. Therefore our aim was to define conditions
suitable for regeneration from explants which can
be taken from adult plants. The publications deal-
ing with shoot regeneration or somatic embryo-
genesis starting from adult plant parts are limited
and often restricted to one model genotype (e.g.
‘Frensham’ KrishnaRaj et al., 1997 or ‘Dubonnet’
Boase et al., 1996). Exceptions are the studies of
Robichon et al. (1997) who analysed seven P.
peltatum cultivars, of Agarwal and Ranu (2000)
with three P. zonale cultivars and that of Haensch
(1999) testing thirty-five cultivars from both P.
zonale and P. peltatum hybrids. In order to assess
the general practicability of the regeneration system
for pelargonium breeding, we performed our study
with sixteen cultivars of economic importance.

The 16 cultivars of the present study differed in
their regeneration ability as well as in the intensity
of regeneration. In total, in eight of them shoot
regeneration from petiole explants was obtained
on medium containing 10 lM TDZ. These differ-
ences in the in vitro response are often observed in
tissue culture studies and thought to be due to the
varying endogenous hormone contents or differing
ability to respond to the growth regulators applied
in the medium. In addition other factors like the
physiological status of the plant in the greenhouse
could have an effect on the regeneration response.
When explants were taken from in vitro grown
shoots of 12 of the cultivars which were tested in
the present study, shoot regeneration was observed
in two genotypes (P08 and P10), which did not
react in the present study using explants from
greenhouse grown plants. Assumed reasons for
this are physiological differences in plants grown
under in vitro conditions or deleterious effects of
the surface sterilization procedure.
Between the three cytokinins tested in the
present study, BA was found to be not suitable for
induction of shoot regeneration (Table 2). TDZ
was more efficient than zeatin, although the shoots
were larger and of good quality on zeatin-con-
taining media. The physiological action of TDZ
was the subject of some studies with pelargonium
(Visser et al., 1992; Gill et al., 1994; Hutchinson
et al., 1996). TDZ was shown to have, or induce,
cytokinin activity resulting in shoot formation in
the present study as well as in previous reports on
other genotypes of P. peltatum (Robichon et al.,
1997). On the other hand, Visser et al. (1992) re-
ported that TDZ also meets the auxin requirement
for inducing somatic embryogenesis on hypocotyl
cultures. However, in the regeneration system used
in the present study, the structures developing on
the callus only had shoot meristems and were
connected to the callus.
One aim of our study was to start from ex-
plants which can be taken from adult plants. Both
explant types derived from different parts of the
leaf gave rise to shoot regeneration in the
responding genotypes. For the majority of culti-
vars tested, petioles gave better regeneration re-
sults than leaf blade explants. These results are in
agreement with the observations made by Robi-
chon et al. (1997) who found higher shoot regen-
eration rates for petioles as compared to leaf
blades in four P. peltatum cultivars. In our studies,
41
the difference between both explant types was less
pronounced when in vitro plantlets were the ex-
plant source (data not shown), indicating that the
lesser response of the leaf blade explants could be
due to greater sensitivity of this tissue to the sur-
face sterilisation procedure.
Since cefotaxim did not have a significant
inhibitory effect on shoot regeneration at the
concentration of 500 mg l)1, it can be applied for
suppression of agrobacteria after co-culture.
Additionally it did not interact with glufosinate
(Table 3).
Glufosinate at the lowest concentration
(2.5 lM) reduced callus formation markedly and
inhibited shoot regeneration totally (Figure 3).
Since the explants did not show strong necrotic
reactions within the first days of culture on 2.5 lM
glufosinate and also callus formation occurred to a
low extent, this concentration seems to be appro-
priate for selection of transgenic cells. In other
reports dealing with Agrobacterium mediated
transformation of pelargonium, kanamycin was
used for selection (Boase et al., 1996, 1998;
KrishnaRaj et al., 1997; Bi et al., 1999).
Transformation experiments
The regeneration protocol developed in this study
was applied in the first transformation experiments
comparing two Agrobacterium strains and differ-
ent co-culture durations. Strain EHA 101 was
more virulent than strain LBA 4404 as could be
seen from the GUS expression in petiole explants
(Table 4). Also Meurer et al. (1998) reported
higher transformation frequencies in soybean
when using EHA 101 compared to LBA 4404. On
the other hand, EHA 101 was more difficult to be
suppressed after co-culture. Therefore, for this
strain, the co-culture duration should be limited to
three days, or other antibiotics or combinations of
different antibiotics should be tested for suppres-
sion of bacterial growth.
GUS expression was most prominent in the
outer cell layers of the petioles and it remains to be
proven if GUS-expressing callus derived from
these layers is stably transformed and if shoots can
be regenerated from the transformed cells. Small
plantlets were regenerated from the calluses and
rooted on glufosinate-containing medium, some
of which showed GUS activity in their leaves.
42
The molecular analysis of these plants is in pro-
gress.
Acknowledgements
The authors are grateful to Prof. Hans-Jörg Jac-
obsen and Dr Heiko Kiesecker for supporting us
with the Agrobacterium strains and the vector,
which originally was kindly provided by Prof. Inge
Broer, and to the pelargonium breeders, Selecta
Klemm, Elsner PAC, and Pelargonien Fischer, for
supplying the plant materials. The scholarship of
the German Academic Exchange Service (DAAD)
for Kamran Kaviani is gratefully appreciated.
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