Plant Cell, Tissue and Organ Culture 67: 1–9, 2001.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

1

Review

Recent advances in Pelargonium in vitro regeneration systems

Jugulam Mithila, Susan J. Murch, Sankaran KrishnaRaj & Praveen K. Saxena∗

Department of Plant Agriculture, University of Guelph, Guelph, Ontario N1G 2W1, Canada
(∗ requests for offprints; Fax: 519-767-0755; E-mail: psaxena@uoguelph.ca)

Received 21 February 2000; accepted in revised form 21 May 2001

Key words: genetic transformation, organogenesis, Pelargonium, shoot-tip culture, somatic embryogenesis,
thidiazuron

Abstract
Recent advances in the development of protocols for in vitro culture and genetic manipulation have provided new
avenues for the development of novel varieties of Pelargonium and for use as model systems for investigating
the factors controlling plant morphogenesis. Optimized techniques of meristem culture have supplemented the
culture indexing methods in commercial greenhouse production resulting in availability of large-scale pathogen
indexed planting material. Currently, technologies are available for the mass in vitro propagation of F1 hybrid
Pelargonium through both organogenesis and somatic embryogenesis. The somatic embryogenesis model system
has allowed researchers to identify critical factors controlling plant morphogenesis in vitro such as regulation
of regeneration by growth regulators, choice of explant and characterization of induction and expression phases
of morphogenesis in Pelargonium. Also, optimization of technologies for genetic transformation of Pelargonium
opened up the possibilities for developing genotypes with novel characters, including resistance to some of the
major diseases. Finally, the development of regeneration systems for Pelargonium spp. has facilitated conventional
crop improvement programs, thereby providing a valuable resource to the horticultural industry.

Abbreviations: ASA – acetylsalicylic acid; Ace-AMP1 – Allium cepa antimicrobial protein; BA – N6 -

benzyladenine; BAP – benzylaminopurine; CPPU – forchlorfenuron; DAP – diaminopurine; ELISA – enzyme
linked immunosorbent assay; GUS – β-glucuronidase; IAA – indoleacetic acid; MS – Murashige and Skoog (me-
dium); NAA – naphthaleneacetic acid; PCR – polymerase chain reaction; RAPD – random amplified polymorphic
DNA; TDZ – N -1,2,3-(thidiazol 5-yl)urea

Geraniums: An important horticultural crop
Pelargonium spp., commonly known as geraniums,
belong to the family Geraniaceae and are among the
most economically important bedding and pot plants
in North America with yearly sales in excess of $100
million (Canadian). Nearly all of the 280 species of
Pelargonium originated from South Africa (Kellen,
1983) and Pelargonium zonale was first introduced to
Europe in 1609 (Craig, 1960; Ewart, 1981). Currently,
North America and Europe are the major producers
and distributors of Pelargonium with a global annual
sale of $700 million. Although there are a large num-
ber of different species in the genus Pelargonium, few
of them possess the traits, which are important for
large scale, commercial production viz. flower color,
types, foliage and growth habits. The commercially
important Pelargonium species have been categor-
ized into four major groups: such as zonal geraniums
(Pelargonium x hortorum), regal pelargonium (Pelar-
gonium x domesticum), ivy geraniums (Pelargonium
peltatum) and scented geraniums (Pelargonium sp.).
Conventionally pelargonium are propagated by ve-
getative cuttings or seeds. The process of taking cut-
tings requires the maintenance of a large number of
stock plants and has led to the spread of several ser-
ious bacterial and fungal diseases (Mastalerz, 1971).
Although seed propagated cultivars of Pelargonium
2
are becoming a popular alternative, they lack certain
horticultural attributes such as semi-double florets and
non-shattering found in the vegetatively propagated
cultivars (Harney, 1982). As well, seeds of Pelar-
gonium are very expensive, approximately $120.00
per 1000 seeds (Stokes Growers Guide, 2000); seeds
of some varieties cost as much as $0.30 each. The
high-value Pelargonium crop, therefore, has the po-
tential to benefit from the large-scale propagation
of pathogen indexed plants through tissue culture
(Laughner, 1993). Additional advantages of in vitro
mass propagation over conventional propagation tech-
niques include: rapid multiplication, lower production
costs, convenience in handling, storage and shipping,
as well as the limited quarantine restrictions.
In vitro culture and propagation
Meristem tip culture and culture indexing
The availability of pathogen indexed planting mater-
ial has significantly increased the commercial value
of asexually propagated ornamentals. Also healthy
planting material is an important prerequisite for en-
suring profitable market. Pelargoniums are susceptible
to diseases such as bacterial blight (Xanthomonas
campestris pv. pelargonii), verticillium wilt (Verticil-
lium dahliae) and botrytis (Botrytis cinerea), that can
cause extensive damage to the crop at all stages of
plant growth and development (Dunbar, 1990). These
pathogens spread during routine cultural practices in
the greenhouse and the symptoms often are not seen
until favorable conditions have appeared (Oglevee,
1993). The chemical control of vascular diseases has
not proven to be effective for disease control and
therefore culture indexing is the most popular method
practiced by commercial propagators to limit disease
infection. In response to these problems, standardized
protocols have been developed through meristem tip
culture for the mass production of pathogen-indexed
planting material in Pelargonium (Horst et al., 1976;
Reuther, 1983).
Meristem tip culture is extremely useful for the
production of pathogen-indexed planting material.
The process of in vitro culture of meristems involves
the aseptic culture of shoot tips (0.1–0.5 mm) with at-
tached leaf primordia (Horst and Klopmeyer, 1993).
Culture indexing is a vigorous testing system com-
bined with intense selection and sanitation procedures
(Horst et al., 1976; Reuther, 1983; Horst and Kl-
opmeyer, 1993). The presence of the pathogen is
routinely detected at different steps in a process that
include culturing of a portion of the sterilized tissue
of mother plant on a suitable media, favorable for
the growth of the pathogen (Tammen, 1961). Vegetat-
ive cuttings are tested three times before entering the
propagation system as pathogen indexed stock (Og-
levee, 1993). The whole process takes a year with
increased production costs. Since it is economically
not viable to culture-index each cutting, multiplica-
tion for commercial propagation is done from selected
plants and culture-indexing ensures clean stock for
further propagation (Oglevee, 1993).
Novel technologies have been developed for patho-
gen detection, such as the Enzyme Linked Immun-
osorbent Assay (ELISA) and colorimetric assays are
available commercially (Oglevee, 1993). However,
these methods are limited in their potential for de-
tection of certain pathogens especially low levels of
infection by Xanthomonas campestris pv. pelargonii
(Oglevee, 1993). Recently, protocols have been stand-
ardized for biological enrichment and PCR ampli-
fication to detect small populations of Xanthomonas
campestris pv. pelargonii, in the tissues of Pelar-
gonium (Sulzinski, 1998). This procedure might have
applications in detecting the low levels of infec-
tions associated with the pathogen (Sulzinski, 1998).
Also the efficacy of CO2 enrichment for inhibition
of growth of Xanthomonas campestris pv. pelargonii
was assessed in Pelargonium x domesticum (Jiao et al.,
1999).
Alternative technologies for mass propagation of
Pelargonium
Alternative technologies can be used for the mass
propagation of plants in vitro and to produce com-
mercial stock through either organogenesis or so-
matic embryogenesis. For example, new cultivars have
been obtained when the genetic stability of the pro-
geny from diverse crosses of Pelargonium cultivars
was achieved through embryo rescue (Cassells et al.,
1995). Organogenesis is the process by which cells
and tissues undergo changes which lead to the pro-
duction of a unipolar structure with either shoot or
root primordium with a distinct vascular connection
to the explant vasculature (Thorpe, 1993). In contrast,
somatic embryogenesis leads to the production of a
bipolar structure containing a root and a shoot axis
and a closed, independent vascular system (Thorpe,
1993). As with many other plants, the first tissue

cultured Pelargonium were developed from callus cul-
tures (Mayer, 1956; Harney, 1982; Stephaniak and
Zenkteler, 1982). De novo shoot organogenesis has
been induced on Pelargonium stem segments cultured
under the sterile and non-sterile conditions (Chen and
Galston, 1967). Also regeneration of callus cultures
from the leaf explants on cytokinin enriched medium
was reported in Pelargonium graveolens (Rao, 1994).
In vivo organogenesis
A novel method of de novo organogenesis was de-
veloped for greenhouse grown zonal geraniums (San-
ago et al., 1995; Murch et al., 1997). Zonal geraniums
(Pelargonium x hortorum) watered with low con-
centrations of the plant growth regulator thidiazuron
(TDZ) formed outgrowths on the root tissues and pro-
lific shoots from the crown (Sanago et al., 1995).
Analysis of the plant tissues revealed the accumula-
tion of stress-related metabolites and metabolic energy
prior to the appearance of the regenerative structures
(Murch et al., 1997). The accumulation of these meta-
bolites in the plant tissue would allow for the main-
tenance of metabolic processes and sustained growth
under imposed stress. In this scenario the occurrence
of regenerative structures in TDZ treated geraniums
may be an adaptive reproductory mechanism to over-
come stress or a byproduct of increased metabolic
activity (Murch et al., 1997).
In vitro organogenesis
Mayer (1956) demonstrated that callus and roots,
but not shoots, could be induced on Pelargonium
stem segments exposed to various concentrations of
auxin, adenine, adenosine, KH2 PO4 and the responses
were enhanced by supplementation of the medium
with various plant extracts. Since these early exper-
iments, pith cells, petioles, anthers and shoot tips
of cutting propagated Pelargonium have been used
for the induction of callus which subsequently dif-
ferentiated into shoots and roots (Chen and Galston,
1967; Pillai and Hildebrandt, 1968; Debergh and
Maene, 1977; Harney, 1982) and extensive efforts
have concentrated on the establishment of optimal
conditions for regeneration. Numerous investigators
have also studied the potential for commercial mi-
cropropagation of cutting-propagated Pelargonium via
shoot organogenesis (Chen and Galston, 1967; Pillai
3
and Hildebrandt, 1968; Abo El-Nil and Hildebrandt,
1971, 1973; Kameya, 1975; Hamdorf, 1976; Horst et
al., 1976; Theiler, 1977; Debergh and Maene, 1977;
Reuther, 1983; Yarrow et al., 1987). Of note were the
studies of Pillai and Hildebrandt (1968) who were the
first to produce viral-indexed plantlets from meristem
cultures.
In general, advances in the induction of organo-
genesis in Pelargonium have been slow since the
requirements for the type and concentration of plant
growth substance used to induce regeneration varies
widely with the cultivar and species (Hammerschlag
and Bottino, 1981). The application of this technique
on a commercial scale has also been hampered by low
survival rates, poor development of the apices at the
initiation stage, limited numbers of de novo shoots
and rapid loss of the capacity for initiation of shoot
organogenesis in callus cultures (Desilets et al., 1993).
In addition, a fairly high proportion of the plants
produced through culture showed leaf abnormalities
as a result of genetic instability from callus-derived
plants (Harney, 1982). However, in scented geraniums
(Pelargonium graveolens) genetic variability in callus
cultures produced stable somaclonal variation (Skirvin
and Janick, 1976).
A variety of factors have been investigated for
the induction of direct shoot organogenesis in Pelar-
gonium. In some cultivars, shoot organogenesis has
been improved by a reduction of the mineral concen-
tration of MS medium (Hildebrandt and Harney, 1988)
and by optimized plant growth regulator concentra-
tions (Desilets et al., 1993). The choice of explant
has also been shown to significantly effect regenera-
tion efficiency. Dunbar and Stephens (1991) reported
protoplast-derived regeneration from callus tissue of
leaf explants in regal pelargonium (P. x domesticum).
De novo shoot organogenesis was induced on intact
seedlings of hybrid P. x hortorum in response to sup-
plementation of the culture medium with BAP (Qure-
shi and Saxena, 1992). More recently, an investigation
of the influences of seedling age, growth regulator
concentration and excision orientation on shoot or-
ganogenesis showed that hypocotyl explants of young
seedlings had the greatest potential for regeneration
(Chang et al., 1996). Plantlet regeneration was also
achieved on petiole sections of ivy pelargonium ex-
posed to a combination of TDZ and IAA in the culture
medium (Robichon et al., 1997). Adventitious shoots
were induced from thin layer hypocotyl explants in P.
x hortorum (Croke and Cassells, 1997). Also, shoot re-
generation was observed in fertilized ovules of a cross
4
between P. x hortorum and P. peltatum, cultured in
vitro (Fukuda et al., 1997). Recently, Cassells et al.
(1997) have established a screening technique for the
detection and selection of somaclonal variations de-
rived from irradiated explants in P. x hortorum using
RAPD-analysis. Together these studies have formed
a solid basis for further studies that will lead to the
eventual development of efficient production systems
for Pelargonium.
Somatic embryogenesis
Somatic embryogenesis – the process by which so-
matic cells develop into differentiated plants through
characteristic embryological stages without fusion of
the gametes (Williams and Maheswaran, 1986) – is an
alternate plant regeneration procedure and may offer
advantages over shoot organogenesis. Somatic embryo
has a single cell origin, is bipolar and is not connected
by the vascular system to the explant and, therefore,
the plants derived from somatic embryos will be true
clones and not chimeras (Ammirato, 1983). Other ad-
vantages of regeneration by somatic embryogenesis
include: an increased efficiency in the formation of
plantlets with fewer steps, with a concomitant re-
duction in labor, time and cost; the potential for the
production of much higher number of plantlets and a
greater morphological and cytological uniformity of
regenerated plantlets (Ammirato, 1983).
In 1979, Cassells noted a few “bipolar vegetative
embryos” in callus cultures of tetraploid zonal Pelar-
gonium cultivar Irene. Marsolais et al. (1991) used
petioles and hypocotyls in their efforts to produce
somatic embryos and achieved success with all 30 dip-
loid and tetraploid cultivars of P. x hortorum and P. x
domesticum tested. Different P. x hortorum cultivars
were found to respond with varying frequency to a
media containing IAA and BAP. Explants of the cul-
tivar Scarlet Orbit Improved were found to have the
greatest frequency of somatic embryogenesis of the
30 genotypes tested with an average of 6.9 somatic
embryos per explant, while the cultivar Ringo Rose
was the least responsive with a mean of 0.2 somatic
embryos per explant (Wilson, 1990). This protocol
was further refined with variations of the media con-
stituents viz. sucrose, ammonium nitrate, auxins and
cytokinins (Wilson et al., 1996). The somatic em-
bryos were found to originate as early as the fourth
day of culture from single subepidermal parenchyma
cells. Development was observed to proceed through
the globular, heart-torpedo, and cotyledonous stages
characteristic of in vivo embryogenesis, at days 8, 14
and 24, respectively and a suspensor-like structure was
observed but was not always present (Wilson et al.,
1994). Haensch (1999) investigated the potential of
23 Pelargonium cultivars for somatic embryo forma-
tion with various media combination and reported that
the response of cultures was largely influenced by the
genotype of the cultivar.
In these experiments and others, various combina-
tions of auxins and cytokinins were shown to induce
somatic embryogenesis in the cultivated Pelargonium
species (Marsolais et al., 1991; Slimmon et al., 1991;
Wilson et al., 1996) but the frequency of embryogen-
esis was quite low. A significant advancement in the
development of protocols for induction of somatic em-
bryogenesis was the discovery of the efficacy of the
plant growth regulator TDZ for induction of regenera-
tion in a wide range of species including Pelargonium
(Murthy et al., 1998). TDZ-induced somatic embryo-
genesis was first described in zonal Pelargonium ex-
plants by Visser et al. (1992). These findings were
particularly interesting since the requirement for both
exogenous cytokinin and auxin was satisfied by the
synthetic cytokinin – like compound TDZ. In previous
research, the presence of an auxin or other auxin-like
compound such as indoleacetic acid and phenylacetic
acid (Marsolais et al., 1991; Slimmon et al., 1991) was
required in combination with the cytokinin BAP for
somatic embryogenesis to occur.
The substitution of TDZ for the IAA-BAP re-
quirement for embryogenic induction demonstrated
that TDZ may possess an auxin-like property or may
impinge upon the endogenous auxins by modifying
their biosynthesis or metabolism (Visser et al., 1992).
Comparison of the physiological and morphological
evaluation of somatic embryogenesis in P. x hortorum
explants exposed to either TDZ or the combination
of auxin and cytokinin indicated that TDZ stimulated
an accumulation of endogenous auxins and cytokinins
in the explants and an increase in the number of
meristematic centers leading to formation of signific-
antly more somatic embryos than that observed on the
auxin-cytokinin treatments (Hutchinson et al., 1996b).
In additional studies, the frequency of somatic em-
bryogenesis was improved by culture of thin layer
explants from the hypocotyls of P. x hortorum seed-
lings (Gill et al., 1993). The thin layer explants consist
of the epidermis and a few subepidermal cell lay-
ers that maintain cell-cell contact comparable to that
found in intact plants and large explants but without

the complex structural and functional relationships
among various types of tissues (Gill et al., 1993).
The efficacy of TDZ for induction of somatic embryo-
genesis was further demonstrated in a wide range of
explants including: intact seedlings (Qureshi and Sax-
ena, 1992), etiolated hypocotyls (Visser et al., 1992;
Hutchinson et al., 1996b) and cotyledons (Singh et al.,
1996). In addition to TDZ, other, similar compounds
such as forchlorfenuron (CPPU) exhibited a similar
capacity for induction of morphogenesis in cotyledon-
ary tissues of the P. x hortorum cultivars Scarlet Orbit
Improved (Singh et al., 1996) and Ringo Rose (Murthy
et al., 1996). Recently investigations with hypocotyl
explants of P. x hortorum have indicated enhanced em-
bryo formation with the addition of smoke saturated
water in TDZ-enriched media (Senaratna et al., 1999).
The TDZ-induced somatic embryos were hydrogel-
encapsulated using 3% sodium alginate and 50 mM
calcium chloride to produce ‘artificial seeds’. These
seeds were found to germinate normally and to grow
into flowering plants within 12–14 weeks (Gill et al.,
1994) thereby demonstrating the potential for com-
mercial production of artificial seed of Pelargonium
via somatic embryogenesis.
One of the most interesting aspects of P. x
hortorum (cv. Ringo Rose) somatic embryogenesis
was the discovery of the modulation of morphogen-
esis by a symbiotic bacterium (Visser-te Nyenhuis et
al., 1994). A growth promoting bacterium was isolated
which induced regeneration on the recalcitrant cultivar
Ringo Rose at a higher frequency than was previ-
ously reported for explants cultured on medium sup-
plemented with various combination of plant growth
regulators. The bacterium was isolated as an in vitro
contaminant from cultures of Pelargonium seedling
explants and was identified to have close homology
to the Bacillus circulans. Co-cultivation of hypocotyl
explants with the bacterium during the first week of in-
duction was found to improve both the frequency and
quality of somatic embryos (Murthy et al., 1999). Also
from this study it has been indicated that among vari-
ous treatments used to disrupt the bacterial cells, only
sonication improved somatic embryogenesis. Promot-
ive effects of sonicated extracts was likely due to the
presence of some live bacterial cells which survived
ultrasonic disruption. Although the mode of action of
the bacterium in the induction of regeneration remains
unclear, these data provide interesting new directions
for further studies of plant–microbe interactions and
may provide the basis for the development of new
5
protocols for the remaining recalcitrant commercial
cultivars.
Pelargonium somatic embryogenesis: A model
system for investigating regulation of plant
morphogenesis
The TDZ-regulated Pelargonium somatic embryogen-
esis system offered a unique opportunity for investig-
ations into basic scientific questions related to the reg-
ulation and expression of morphogenesis in a higher
plant. A standardized protocol was developed for these
studies in which the Pelargonium hypocotyl sections
were exposed to a relatively high concentration of
TDZ (20 µM) for a 3-day induction period followed
by subculture onto a basal medium for an 18-day
expression period (Hutchinson and Saxena, 1996a).
Since the experimental system was regulated by a
single growth regulator it was possible to investig-
ate several aspects in the regulation of morphogenic
responses.
The observation that TDZ induced somatic em-
bryogenesis in the absence of other growth regulating
compounds led to the hypothesis that the modulation
of endogenous auxin may be one of the mechanisms
involved in TDZ-induced embryogenesis (Hutchin-
son et al., 1996a). In general, presence of auxins
or auxin-like substances is required for the induction
and proliferation of cells that later differentiate into
somatic embryos. Both auxin transport and auxin-
action inhibitors were found to significantly reduce
the number of somatic embryos formed in response
to TDZ (Hutchinson et al., 1996a). It was interesting
to note that while the auxin-action inhibitor reduced
the endogenous concentration of IAA, the auxin trans-
port inhibitor actually increased the endogenous IAA
concentration but decreased the regenerative response
(Hutchinson et al., 1996a). Together these data provide
evidence that both the concentration and transport of
endogenous auxin are closely tied with the induction
and development of somatic embryos in Pelargonium.
Cytokinins are another group of plant growth reg-
ulators that are closely associated to TDZ-induced so-
matic embryogenesis. Supplementation of the culture
medium with TDZ was found to increase the con-
centration of endogenous cytokinins in Pelargonium
hypocotyls (Hutchinson and Saxena, 1996a). In ad-
dition, the inhibitor of cytokinin action, diaminop-
urine (DAP) significantly decreased the regenerative
response but addition of adenine sulphate reversed the
6
DAP-mediated inhibition of embryogenesis thereby
confirming the requirement for the accumulation of
cytokinin for induction and expression of somatic
embryogenesis (Hutchinson and Saxena, 1996a).
In contrast, the presence of gibberellic acid during
either the induction or expression phases of Pelar-
gonium somatic embryogenesis was found to signific-
antly reduce the number of somatic embryos formed
on each explant (Hutchinson et al., 1997a). As well,
TDZ-induced accumulations of ethylene in the head-
space of Pelargonium cultures were found to signi-
ficantly decrease the morphogenic potential of the
Pelargonium explants (Hutchinson et al., 1997b).
Acetylsalicylic acid (ASA), a regulatory molecule
in the signal transduction pathways has been shown
to stimulate a variety of physiological reactions viz.
thermogenesis, flowering, protoplast division and in-
hibition of ethylene biosynthesis (Raskin, 1992). Sup-
plementation of the TDZ medium with ASA signi-
ficantly increased the number of somatic embryos
that developed on each explant (Hutchinson and Sax-
ena, 1996b). It was equally interesting that the ap-
plication of ASA synchronized the development of
TDZ-induced somatic embryos such that more than
80% of the somatic embryos were at the cotyledon-
ary stage of development at day 35 of the culture
experiment (Hutchinson and Saxena, 1996b). In sub-
sequent studies, exposure to TDZ was found to induce
an accumulation of ethylene in the headspace of the
culture vessels and the addition of ASA to the TDZ
medium significantly reduced this ethylene accumula-
tion (Hutchinson et al., 1997b). These data suggested
the possibility that the TDZ-induced embryogenesis
is inhibited by increased levels of ethylene accumu-
lation.
Genetic engineering of Pelargoniums
There have been several recent reports of genetic
transformation of Pelargonium cultivars for the devel-
opment of value-added crops. In 1994, Pellegrines-
che et al. reported the development of an in vivo
protocol for genetic transformation of rooted cut-
tings of lemon-scented geraniums with Agrobacterium
rhizogenes. Robichon et al. (1995) were the first
to report the production of transgenic Pelargonium
x hortorum via Agrobacterium tumefaciens. These
two reports provided the foundation for the devel-
opment of a system for somatic embryogenesis and
Agrobacterium-mediated transformation for scented
geraniums (Pelargonium sp. ‘Frensham’) (Krishna-
Raj et al., 1997). The protocol developed in this
study was unique in that it coupled high-frequency
somatic embryogenesis with efficient gene insertion
and expression for the production of relatively large
numbers of transgenic plants (KrishnaRaj et al., 1997).
In subsequent studies, this protocol was used to pro-
duce transgenic plants containing a gene encoding
for the antimicrobial protein Ace-AMP1. The incor-
poration of this gene into Pelargonium sp. ‘Fren-
sham’ conferred an increased resistance to infection
by Botrytis cinerea (Bi et al., 1999). Pelargonium
cultivars with relative levels of resistance to Botrytis
have recently been identified and it has been repor-
ted that diploid genotypes possess greater resistance
than tetraploid cultivars (Uchneat, 1999). These gen-
otypes may provide additional genetic information for
incorporation into resistance breeding programs for
Pelargonium. An improved protocol for genetic trans-
formation of P. x domesticum was reported using acet-
osyringone to induce vir genes on the Agrobacterium
tumefaciens Ti plasmid (Boase et al., 1998).
Conclusions and future prospects
Significant progress has been made in in vitro re-
generation systems in various types of Pelargonium.
As well, a Pelargonium in vitro model system has
been used for the elucidation of many of the factors
controlling plant morphogenesis. Nevertheless, the de-
velopment of regeneration systems for the formation
of somatic embryos in liquid medium has not been
accomplished. This type of system would be desirable
as there is potential for commercial scale artificial seed
production. As well, protocols for microspore and pro-
toplast regeneration and breeding via somatic hybrid-
ization remain to be optimized for various genotypes
of Pelargonium.
The findings of the in vitro regeneration studies
have also resulted in the development of genetic trans-
formation systems for gene transfer in Pelargonium.
The wide genetic diversity in Pelargonium sp. opens
immense perspectives to improve commercially im-
portant traits such as novel flower color, early and
continued flowering. Studies have indicated that ge-
netically controlled biochemical reactions that occur
in the apical meristem can induce early flowering in
Pelargonium (Craig, 1983). The genetic transforma-
tion systems that have been developed in other or-
namentals such as in petunias and chrysanthemums

(Deroles et al., 1997) for the production of rare flower
color, might be applicable for Pelargonium. The de-
velopment and optimization of biotechnological ap-
proaches has opened up some additional possibilit-
ies for further research in Pelargonium including the
development of cultivars resistant to diseases, espe-
cially to bacterial infections caused by Xanthomonas
campestris pv. pelargonii, through cell cultures, in-
duced mutations and genetic transformation and ap-
plication of techniques such as in vitro fertilization and
embryo rescue could be directed toward elimination
of reproductive barriers, thereby increasing the overall
genetic pool with additional germplasm for desirable
characters.
Acknowledgements
The financial support of the Natural Sciences and En-
gineering Research Council of Canada is gratefully
acknowledged.
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