JBA-06688; No of Pages 14

Biotechnology Advances xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Gerbera micropropagation
Jean C. Cardoso a,⁎, Jaime A. Teixeira da Silva b,c
a
Depto. de Desenvolvimento Rural, Centro de Ciências Agrárias/Universidade Federal de São Carlos (CCA/UFSCAR), Rod. Anhanguera, km 174, CEP 13600-000 Araras, Brazil
b
Department of Horticultural Science, Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken 761-0795, Japan
c
P. O. Box 7, Miki-cho post office, Ikenobe 3011-2, Kagawa-ken 761-0799, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Gerbera jamesonii (gerbera) is an important cut-flower in the global floricultural industry. Micropropagation
Received 17 October 2012 is the main system used to clonally propagate gerbera in vitro resulting in the production of millions of plant-
Received in revised form 9 May 2013 lets each year. Numerous types of explants and protocols for micropropagation have been established and
Accepted 26 May 2013
used for gerbera. Shoot tips are the commonly used explant while adventitious shoot induction from the ca-
Available online xxxx
pitulum is also a popular method. Most papers in the literature have focused on testing the influence of dif-
Keywords:
ferent types and combinations of plant growth regulators with the aim of improving the regeneration and
Gerbera jamesonii multiplication stage of one or few cultivars. Genotype is one of the most influential factors on the response
Clonal propagation of gerbera in vitro. Despite this, no successful universal protocol has yet been developed for multiple cultivars,
Culture medium limiting the usefulness of current protocols for commercial biotechnology labs. Slow-growing endogenous
Explants bacteria are one of the most important problems in gerbera micropropagation but require more studies on
Adventitious shoot induction control and prevention. Individual shoots are normally easy to root, usually in excess of 90% of plantlets,
Genotypes but the acclimatization stage requires improvements and new technologies to increase the survival of plants.
Acclimatization
Epigenetic variations in micropropagated gerbera are frequently observed only with high concentrations of
Somaclonal variations
cytokinins in the culture medium but somaclonal variation is rare.
© 2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction and history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2. Propagation and micropropagation of gerbera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.1. In vitro establishment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.1.1. Shoot tip culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.1.2. Organogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.1.3. Cell suspension culture and somatic embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.1.4. Factors that affect in vitro establishment and organogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2. Multiplication stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2.1. Factors that affect shoot multiplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.3. Rooting and elongation stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.3.1. Factors that affect rooting of gerbera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.4. Acclimatization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.5. Photoautotrophic culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3. Genetic and epigenetic variations in micropropagation of gerbera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Abbreviations: ADS, adenine sulphate; Aux, auxin; BA, 6-benzyladenine; B5, Gamborg et al. 1968; Ck, cytokinin; DKW, Driver and Kiniyuki 1984; IAA, indole-3-acetic acid; IBA,
indole-3-butyric acid; 2-ip, 2-isopentenyladenine; ISSR, inter-simple sequence repeat; Kin, 6-furfurylaminopurine (kinetin); LS, Linsmaier and Skoog 1965; MS, Murashige and
Skoog 1962; ½ MS, half-strength Murashige and Skoog (1962); NAA, 1-naphthaleneacetic acid; N6, Chu 1978; PGR, plant growth regulator; Phot, photoperiod; PPFD, photosynthet-
ic photon flux density; RAPD, random amplified polymorphic DNA; RFLP, restriction fragment length polymorphism; SE, somatic embryogenesis; TDZ, thidiazuron; tTCL, transverse
thin cell layer; Zea, Zeatin.
⁎ Corresponding author. Tel.: +55 1997646233.
E-mail addresses: jeancardosoctv@gmail.com (J.C. Cardoso), jaimetex@yahoo.com (J.A. Teixeira da Silva).

0734-9750/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.biotechadv.2013.05.008

Please cite this article as: Cardoso JC, Teixeira da Silva JA, Gerbera micropropagation, Biotechnology Advances (2013), http://dx.doi.org/10.1016/
j.biotechadv.2013.05.008
2 J.C. Cardoso, J.A. Teixeira da Silva / Biotechnology Advances xxx (2013) xxx–xxx
1. Introduction and history
The Gerbera complex belongs to the Mutisiinae (tribe Mutisieae)
and includes around 100 species distributed in seven genera, of
which Gerbera has approximately 29 (Katinas, 2004) to 37 (Gao and
Hind, 2011) species, spanning from Africa to Asia (Hansen, 1988;
Katinas, 2004), although there is heated discussion about the number
of species in this genus complex (Hansen, 2006; Katinas, 2004;
Nesom, 2004). Gerbera jamesonii, better known by its popular name,
gerbera, belongs to the Asteraceae (Compositae).
The genus was named Gerbera after a German naturalist, Traugott
Gerber although it was the Scottsman Robert Jameson who discovered
the species in about 1880 near Barberton in the former Transvaal, in
South Africa. At that time, he donated the plant to the Durban Botanical
Garden where John Medley Wood, the curator there, sent the plant to
Harry Bolus for identification of the species. Harry Bolus then sent the
species to the Royal Botanic Gardens in Kew, UK, suggesting the scien-
tific name as G. jamesonii. As the first official description of the species
was made by J. D. Hooker and published in 1889 by Curtis Botanical
Magazine, it became officially registered thereafter as G. jamesonii
Bolus ex. Hook f. (University of Helsinki, 2012).
A programme of genetic improvement of gerbera was initiated
about ten years after its discovery, most likely by the Englishman
Richard Irwin Lynch, who started an inter- and intraspecific hybridiza-
tion programme (G. jamesonii × Gerbera viridifolia) (Seifert, 2011),
eventually developing the actual current cultivars that are used for
pot plants and cut flowers.
In fact, it is the dedicated focus to genetic improvement to obtain
new cultivars, particularly by commercial companies based in the
Netherlands, as well as the fact that this ornamental grows very
well in greenhouse culture in most countries around the world, that
rapidly led to the development of new cultivars, making it one of
the ornamental market leaders (Bhatia et al., 2011; Chakabrarty and
Datta, 2008). These breeding companies introduced many new culti-
vars with different inflorescence colors every year into the world
market, but failed to develop resistant cultivars to important pests
and diseases, which is actually one of the major problems in gerbera
culture. Increasing pressure to reduce the use of agrochemicals in ag-
riculture and horticulture, including in ornamental plant culture, is a
priority, but to achieve this goal, needs resistant cultivars.
The inflorescences of commercial gerbera varieties are renowned for
their array of bright colors that serve a small, but distinct slice of the glob-
al ornamental trade, either as potted flowering plants, or as cut flowers. It
is not easy to obtain market data. In the USA, in 2010, the wholesale
value of the cut flower industry was U$375 million, of which gerbera
daisies accounted for 32.7 million, or 8.7% (NASS-USDA, 2011).
Cut-flowers in general, such as roses and gerberas, actually repre-
sent an important social improvement to many developed and devel-
oping countries around the world, increasing and improving the
quality of life. According to Dolan et al. (2002) and Muhammad et
al. (2010), the cut flower production for export provides jobs, in-
creases trade, provides education, health and child care services,
and social development. At the same time, the problems of labor con-
ditions in the cut flower industry and trade in developing countries
persists, but with international pressure and code implementation
for the sector, there are signs of some improvements in these condi-
tions. As an example, Hale and Opondo (2005) studied the case
of the Kenya–UK cut flower supply chain. Kenya is an important
example of floriculture representing an important tool to social and
economic development in developing countries (Hale and Opondo,
2005).
2. Propagation and micropropagation of gerbera
Gerbera can be traditionally propagated by seed (sexual reproduc-
tion) or by vegetative propagation using stem cuttings, or division of
Please cite this article as: Cardoso JC, Teixeira da Silva JA, Gerbera micropropagation, Biotechnology Advances (2013), http://dx.doi.org/10.1016/
j.biotechadv.2013.05.008
the rhizome in vivo (Leffring, 1971; Osiecki, 1988; Son et al., 2011), or
by in vitro micropropagation (Shabanpour et al., 2011), although it is
only the latter method that provides a reliable method for the clonal
propagation or elite germplasm with apparent relative genetic fideli-
ty (Kanwar and Kumar, 2008).
The propagation of gerbera by seed is possible in some gerbera
cultivars, although the greatest problem with such a method is the
high level of heterozygosity, which can be problematic for the cut
flower trade that requires coordinated flowering of uniform size and
color (Harding et al., 1991). Despite these risks, the commercialized
gerbera for use as pot plants has primarily relied on seed (Ludwig et
al., 2008, 2010). Moreover, in some countries, seeds of superior culti-
vars are used to initiate in vitro cultures for micropropagation pur-
poses (Altaf et al., 2009; Budi, 2000; Feng et al., 2009), primarily
due to the ease of this technique and not necessarily intimidated by
payment of royalties to breeders, even though the high genetic vari-
ability of plantlets derived from seeds remains the greatest limitation
of this technique.
One of the greatest weaknesses of using traditional vegetative prop-
agation by splitting or division of rhizomes or clumps is the low rate
of propagation (Kanwar and Kumar, 2008; Son et al., 2011), the long pe-
riod of time required to obtain commercial quantities of new plants,
around five plants from one per year (Kumar and Kanwar, 2007) and
the increase in the frequency of plantlets with phytosanitary problems
(Das and Singh, 1989), mainly due to the use of non-sterile tools at
the time of cutting and division of parts of rhizomes or clumps.
In fact, micropropagation using terminal buds/apices or through
organogenesis of somatic tissue is considered to be the only possible vi-
able method for the rapid mass propagation of elite gerbera germplasm
(Bhatia et al., 2009) while maintaining the genetic fidelity (i.e., reducing
genetic and epigenetic variation) of these cultivars (Bhatia et al., 2009,
2011; Cardoso and Teixeira da Silva, 2012; Reynoird et al., 1993). More-
over, plant tissue culture methods such as shoot tip culture result in
disease-free plantlets (Dobránzki and Teixeira da Silva, 2010), including
floricultural and ornamental plants such as chrysanthemum (Teixeira
da Silva, 2003) and others (Rout et al., 2006). Such micropropagation
protocols should be developed within a wider genetic improvement
programme aimed at creating disease-free germplasm.
The induction and regeneration of micropropagated gerbera plant-
lets has been extensively studied (Table 1). Plantlets have been
obtained from several kinds of tissues, including the culture of shoot
tips (Cardoso and Teixeira da Silva, 2012; Huang and Chu, 1985;
Murashige et al., 1974), axillary buds (Murashige et al., 1974), leaves
(Aswath and Choudhary, 2002a; Palai et al., 1998; Radojević et al.
1987), petioles (Orlikowska et al., 1999), flower buds (Chakabrarty
and Datta, 2008; Pierik et al., 1973, 1975; Posada et al., 1999; Son et
al., 2011), capitulum (Pierik et al., 1975; Shabanpour et al., 2011;
Topoonyanont and Dillen, 1988) and ovules (Meynet and Sibi, 1984;
Miyoshi and Asakura, 1996; Tosca et al., 1999).
Pierik et al. (1979) concluded that shoot tip culture is more suit-
able for gerbera mass propagation than capitulum organogenesis,
but Murashige et al. (1974) reported advantages and disadvantages
of both techniques and observed that shoot tip culture is more
rapid, but needs a high number of explants for the establishment
stage, because of the high contamination of explants (around 80%).
Capitulum as an explant results in fewer shoots per explant, but the
contamination rate is very low (10%). Shailaja (2002) agreed that
bud flowers and inflorescences were the best explants to initiate ger-
bera micropropagation, but observed that each cultivar needed a dif-
ferent protocol to improve shoot induction and regeneration. Tyagi
and Kothari (2004) compared organogenesis from leaves and capitu-
lum in vitro and obtained shoot regeneration in both, although the
highest number of shoots/explant was obtained from the capitulum
(10/explant) rather than from leaf explants (6.8).
Another in vitro system of propagation was successfully developed
for gerbera using 0.2–0.5 mm thick receptacle explants as transverse
 J.C. Cardoso, J.A. Teixeira da Silva / Biotechnology Advances xxx (2013) xxx–xxx 3

Table 1
Type of explant, experimental purpose and of adventitious shoot regeneration in the establishment stage of Gerbera jamesonii cultivars.

Gerbera jamesonii cultivar Type of explant Experimental purpose Advent shoot regeneration References

Orange and Pink Cap Genotypes Direct Shabanpour et al. (2011)

Pink MS salts Direct Shabanpour et al. (2011)
Orange and Pink PGRs Direct ? Shabanpour et al. (2011)
Jaguar Cream Cap Indirect Rezende et al. (2008)
Arianna, Bonnie, Tobia FB PGRs Direct ? Son et al. (2011)
Sunglow LSeg PGRs Indirect Shabbir et al. (2012)
PGRs Indirect Shabbir et al. (2012)
Cabana ShT, Cap Direct Bhatia et al. (2009)
Leaf-callus Indirect Bhatia et al. (2009)
Not described CapSeg Direct Chakabrarty and Datta (2008)
ShT PGRs Direct
AL101 ShT Direct and indirect Cardoso and Teixeira da Silva (2012)
Jaimy and Mammut Young Cap PGRs Direct Rabori and Ghazvini (2009)
Ten tTCL Genotype Direct and Indirect Nhut et al. (2007)
Interspecific Culture media Direct Nhut et al. (2007)
crosses Flower bud age Direct and indirect Nhut et al. (2007)
Position of tTCLs Direct and indirect Nhut et al. (2007)
Sciella (Yellow flowers) ShT PGRs Direct Gantait et al. (2010)
Not described Pet PGRs Direct ? Hasbullah et al. (2008)
Salvador, Goliath and Zingaro Young Cap Genotypes Direct Misra et al. (2010)
Dana Ellen, Rosaline, Silvester and Sunway Young Ped Genotypes Indirect Misra et al. (2010)
Red colour cut Pet PGRs and Expl Indirect Kumar and Kanwar (2006)
Flower gerbera CA from Pet PGRs Indirect Kumar and Kanwar (2006)
Not described IVS PGRs Direct Naz et al. (2012)
IVS PGRs Direct Naz et al. (2012)

Adv—adventitious; CA—callus; Cap—capitulum; CapSeg—capitulum segment; FB—flower bud; IVS—in vitro shoot; LSeg—leaf segment; Ped—peduncle; Pet—petiole; ShT—shoot tip;
tTCL—transverse thin cell layer culture.

thin cell layers or tTCLs (Nhut et al., 2007). These authors investigated
the influence of several factors and observed that the percentage
of callus induction and shoot regeneration, as well as the number
of shoots/explant, were strongly influenced by genotype, having
obtained 57–95% shoot regeneration in 10 genotypes on MS medium
(Murashige and Skoog, 1962) supplemented with 3% sucrose,
0.02 mg L−1 thidiazuron (TDZ), 0.8 mg L−1 adenine sulphate (ADS)
and 10% (v/v) coconut water (CW). This medium was found to be
best for shoot regeneration (92–95%) and number of shoots/explant
(4.4–5.1). Nhut et al. (2007) also observed that 10-d old flower buds,
rather than 7 and 14-d, was the best for receptacle tTCL culture, with
91% of explants forming shoots with 4.3 shoots/explant. 7-d old explants
promoted high callus induction, but least shoots. Moreover, these au-
thors observed that explants from middle layers of the receptacle, rather
than exterior layers, were the best to directly induce shoot regeneration
(94–100%) and maximum number of shoots/explant (3.1–4.2). TCLs are
important tools in plant micropropagation (Teixeira da Silva, 2013;
Teixeira da Silva and Dobránszki, 2013).
Micropropagation of gerbera has also encompassed other objec-
tives that exceed simple propagation such as conservation (Meyer
and Van Staden, 1988), inflorescence color genetics (Teeri et al.,
2006), physiology related to the biosynthesis of secondary metabo-
lites (Chakabrarty and Datta, 2008; Elomaa et al., 2003) and the
acquisition of induced thermotolerance (Marmiroli et al., 1997),
breeding through induced mutations (Jerzy and Lubomski, 1992;
Laneri et al., 1990), or the induction of autotetraploids (Li et al.,
2009) and haploids (Miyoshi and Asakura, 1996; Tosca et al., 1999),
and finally providing a regeneration basis for developing transgenic
plants (Elooma et al., 1993; Korbin et al., 2002; Nowak et al., 1997)
(Table 2).
2.1. In vitro establishment
2.1.1. Shoot tip culture
The shoot tip culture of gerbera is the most rapid form of clonal
propagation and is the most trusted technique to obtain plantlets
from micropropagation without somaclonal variation (genetic and
epigenetic changes obtained through clonal propagation) (Fig. 1).
The use of shoot tip culture is made all the more difficult because of
problems associated with medium contamination (Fig. 1B), particu-
larly by endogenous bacteria (Fig. 1D) that take hold following inoc-
ulation of culture media (Altaf et al., 2009; Cardoso and Teixeira da
Silva, 2012; Murashige et al., 1974). This difficulty is implicitly ob-
served in the literature since there has been a gradual increase in
the use of other explants such as leaves or capitulum tissues
(Shabanpour et al., 2011) to replace shoot tip culture (Table 1), with-
out any scientific reason or explanation being provided by re-
searchers conducting these studies as to the logical choice of these
alternative explants. Only one study have openly, frankly and realisti-
cally shown the difficulty and seriousness of such contamination
(Cardoso and Teixeira da Silva, 2012). Part of the problem lies in the
lack of willingness of most top-tier international plant science
journals to address (= publish) these issues for fear of criticism.
Fifteen strains of ‘slow-growing’ bacteria were identified and isolated
from gerbera shoots, seven of which were identified as being
of the genus Bacillus, five being Coryneforms and two identified as
Pseudomonas fluorescens and P. putida. Through antibiograms, all bac-
teria were susceptible to doxyclicline, but the antibiotic only acted
bacteriostatically (Podwyszynska and Hempel, 1987). No other stud-
ies in the gerbera literature address these issues. The excision of the
leaves combined with greenhouse growth of mother plants, rather
than plants from the field, and use of shoot tips from new sprouts
(Fig. 1A) is one strategy to reduce microorganism contamination in
shoot tip culture (Cardoso and Teixeira da Silva, 2012; Van de Raalte,
1978) (Fig. 1C).
Despite these limitations, some gerbera micropropagation proto-
cols have been successfully established (Table 1, Fig. 1). Huang and
Chu (1985) described a method of shoot tip culture for gerbera
using semi-solid half-strength (½ MS) with 2% sucrose, 5 mg L−1 of
6-benzyladenine (BA) and 0.1 mg L−1 of indole-3-acetic acid (IAA).
Cardoso and Teixeira da Silva (2012) used MS medium with 3% su-
crose, but reduced the BA concentration to 0.5 mg L−1 and replaced
IAA by 1-naphthaleneacetic acid (NAA) (0.01 mg L−1). In the latter
study, 20–30% of shoots from shoot tip culture were infection-free,
which is ironically high (relative to the published literature). This
percentage of infection-free explants was similar to that obtained by

Please cite this article as: Cardoso JC, Teixeira da Silva JA, Gerbera micropropagation, Biotechnology Advances (2013), http://dx.doi.org/10.1016/
j.biotechadv.2013.05.008
4 J.C. Cardoso, J.A. Teixeira da Silva / Biotechnology Advances xxx (2013) xxx–xxx

Table 2
Main types of explants, experimental purposes and culture media used in other biotechnological purposes rather than propagation in different gerbera species and cultivars.

Species or cultivar Type of explant Experim purpose Culture media Reference

Gerbera aurantiaca Seeds Cons MS Meyer and Van Staden (1988)

Cap Prop MS + 1.1 mg L−1 BA Meyer and Van Staden (1988)
Not-described Pet Mut MS + 2.0 mg L−1 BA + 0.5 mg L−1 NAA Hasbullah et al. (2012)
IVS Mut MS + 2.0 mg L−1 BA + 0.5 mg L−1 NAA Hasbullah et al. (2012)
LF Seg Mut MS + 1.0 mg L−1 BA + 2.0 mg L−1 2,4-D Hasbullah et al. (2012)
86122, 87274, UM Ovules Hap MS + 0.20 mg L−1 BA + 0.1 mg L−1 IAA Tosca et al. (1999)
23-6 and 24-1 CA Ovules Hap MS + 2.0 mg L−1 BA + 0.1 mg L−1 IAA Tosca et al. (1999)
Seventheen UM ovules Hap Ahmim and Vieth (1986) medium (AV) + 0.20 mg L−1 BA + 0.1 mg L−1 IAA Miyoshi and Asakura (1996)
genotypes UM ovules Hap AV (1986) medium + 0.20 mg L−1 BA + 0.1 mg L−1 NAA Miyoshi and Asakura (1996)
CA ovules Hap AV (1986) medium + 80 mg L−1 AS + 2.0 mg L−1 BA + 0.1 mg L−1 NAA Miyoshi and Asakura (1996)
Not-described CSC leaves PGRs MS liquid medium + 1.5 mg L−1 2,4-D Kumar and Kanwar (2007)
PGRs MS + 3.0 mg L−1 BA Kumar and Kanwar (2007)
Terra Regina Pet Seg Tranf MS + 1.0 mg L−1 BA + 1.0 mg L−1 zeatin + 0.1 mg L−1 IAA Elooma et al. (1993)
Sciella shoots Autotetr MS + 8.8 μmol L−1 BA + 20–25 mg L−1 ADS Gantait et al. (2011)
Alaska shoots Prop MS + 3.0 mg L−1 Kin + 0.1 mg L−1 IAA Korbin et al. (2002)
Paul Prince LF, S, bases S Transf LB + 100 μM ACS Korbin et al. (2002)
Zuzzana Co-cult MS (1% SCR) + 1–1.5 mg L−1 TDZ + 0.2–0.5 mg L−1 IAA + 200 μM ACS Korbin et al. (2002)
CA ind MS (3% SCR) + 0.02 mg L−1 TDZ and IAA each + 250 mg L−1 Carb + 2 mg L−1 Kan Korbin et al. (2002)
Reg MS + 1 mg L−1 TDZ, Kan enhanced gradually (final concentration 7 mg L−1) Korbin et al. (2002)

2;4-D—2;4-dichlorophenoxiacetic acid; ADS—adenine sulphate; Autotetr—autotetraploid; BA —6-benzyladenine; CA—callus; Cap—capitulum; Carb—carbecillin; Co-cult—
co-cultivation; Cons—conservation; CSC—cell suspension culture; Hap—haploid; ind—induction; IAA—indole-3-acetic acid; IBA—indole-3-butyric acid; IVS—in vitro shoot; Kan—
kanamycin; Kin—kinetine; LB—lisogeny broth medium; LF—leaf ; MS—Murashige and Skoog (1962) culture medium; Mut—mutation; NAA—naphtaleneaceticacid; Pet—petiole;
PGR—plant growth regulator; Prop—propagation; Reg—regeneration; S—shoot; SCR—sucrose; Seg—segment; TDZ—tidiazuron; Transf—transformation; UM—unfertilized mature.

Murashige et al. (1974). Son (2007) also observed a high percentage
of explant contamination (90%) when using shoot tip (1.0 cm in
length) culture.
The high level of contamination of explants from shoot tips other
than capitulum explants is probably mainly caused by the proximity
of this type of explant with the soil or organic substrate, as demon-
strated for other plant species (Ziv and Lilien-Kipnis, 2000). Conse-
quently, the cultivation of donor plants under hydroponic, mineral
or synthetic substrates can be an alternative to reduce the contamina-
tion of shoot tip explants in gerbera micropropagation (Cardoso,
unpublished results). Such a technique was used by Cardoso and
Teixeira da Silva (2012) and resulted in some explants without bacte-
rial contamination.
2.1.2. Organogenesis
Direct and indirect adventitious shoot induction are both used in
gerbera micropropagation (Table 1). Indirect organogenesis and cal-
lus culture from different kinds of tissues were developed and used
in gerbera micropropagation (Kanwar and Kumar, 2008). Aswath
and Choudhary (2002a) obtained high leaf-derived-callus induction
(65–90%), shoot regeneration frequency from callus (60–83%) and
number of shoots per callus cluster (6–14) in gerbera cv. ‘AV101’
and ‘AV108’. In gerbera, different types of callus were observed from
young leaves growing in vitro (Paduchury et al., 2010; Shabbir et al.,
2012). Shabbir et al. (2012) observed three types of callus, depending
on the plant growth regulator (PGR) used in the medium: friable on me-
dium with IBA (1.0–3.0 mg L−1), friable and nodular on media with
NAA or 2,4-dichlorophenoxyacetic acid (2,4-D) (1.0–3.0 mg L−1), and
compact and nodular on medium with BA (1.0–4.0 mg L−1). In general,
the regeneration of plantlets from callus is valuable for gerbera breed-
ing, and has been used for inducing mutagenesis and to develop genetic
transformation protocols (Elooma et al., 1993; Nowak et al., 1997), al-
though variation in RFLP (restriction fragment length polymorphism)
banding pattern in cv. ‘Cabana’ was observed during micropropagation
(Bhatia et al., 2009), suggesting some level of somaclonal variation.
Such an outcome is problematic when the primary objective is the
propagation of true-to-type genotypes or the development of a breed-
ing system (transgenic) (Table 2) where interest lies only in the intro-
duction or modification of one or few genes while maintaining the
phenotypic and genetic integrity of all other characteristics of a cultivar
(Korbin et al., 2002). Unlike callus culture, direct organogenesis from
different types of explants results in true-to-type plants in most cases
(Bhatia et al., 2009). Capitulum-derived explants are used in most
protocols because they are easier to sterilize than shoot tip culture
(Murashige et al., 1974; Pierik et al., 1982) and because plantlets regen-
erate rapidly (Table 1).
2.1.3. Cell suspension culture and somatic embryogenesis
In gerbera, limited papers have been dedicated to studying cell
suspension culture and the regeneration of plantlets through somatic
embryogenesis (SE). Kumar and Kanwar (2005) regenerated cv. ‘Diablo’
by organogenesis using callus and cell suspension culture from disc and
ray florets, or from petal explants using a three-phase culture, the first
employing MS medium with 2.0 mg L−1 NAA or 2,4-D to induce callus,
followed by callus proliferation and cell suspension culture in liquid MS
medium with 2.0 mg L−1 2,4-D. Callus differentiated into adventitious
shoots and regenerated plantlets in semi-solid MS medium with
2.0 mg L−1 BA and 0.5 mg L−1 IAA (Kumar and Kanwar, 2005).
SE from cell suspension culture of leaf-derived callus was obtained
by Sharma and Srivastava (2005) in cv. ‘Alsmeera’ and by Hasbullah
et al. (2011) using leaves from in vitro germinated seeds, although
the cultivar used was ironically not described (Table 1). Sharma and
Srivastava (2005) obtained SE from cell suspension growth in MS me-
dium supplemented with 0.5 mg L−1 BA and 1.0 mg L−1 NAA.
Hasbullah et al. (2011) used MS medium with 2,4-D (1.8–2.0 mg L−1)
and could induce callus in 100% of cultures, and creamy friable-type cal-
lus was used to proliferate callus and cell suspension cultures in liquid MS
medium with 1.0 mg L−1 2,4-D, 0.1 mg L−1 NAA and 5.0 mg L−1 L-pro-
line. The best differentiation of SE, i.e., 91.3% globular, 81.2% heart, 59.6%
torpedo and 48.3% cotyledonary-stage, were obtained from cell suspen-
sion cultures in semi-solid MS medium containing 1.0 mg L−1 2,4-D,
0.1 mg L−1 NAA and 5 mg L−1 L-proline (Hasbullah et al., 2011).
2.1.4. Factors that affect in vitro establishment and organogenesis
2.1.4.1. Genotype. Genotype is one of the key factors affecting the suc-
cess of in vitro development of gerbera. Son et al. (2011) observed dif-
ferent responses of gerbera cultivars to different BA concentrations
and types of auxins added to the media. In their study, when IAA
(0.1 mg L−1) was used, cv. ‘Arianna’ responded only to 3 mg L−1
BA, ‘Bonnie’ to 5 mg L−1 and ‘Tobia’ to 10 mg L−1 in order for shoots
to regenerate. However, when IAA was replaced by 0.1 mg L-1 NAA,
all cultivars responded to 1.0 mg L−1 BA, although the highest num-
ber of shoots/explant, fresh and dry weight of shoots were obtained

Please cite this article as: Cardoso JC, Teixeira da Silva JA, Gerbera micropropagation, Biotechnology Advances (2013), http://dx.doi.org/10.1016/
j.biotechadv.2013.05.008
 J.C. Cardoso, J.A. Teixeira da Silva / Biotechnology Advances xxx (2013) xxx–xxx 5

A C D

E G
F

H I J K

L M
Fig. 1. Micropropagation of gerbera using shoot tip explants. A. Rhizomes of gerbera donor plants with young shoots after 30-d of cultivation in coconut fiber. B. Isolated shoot tip
that died, with fungal and bacterial contamination. C. In vitro development of shoot tip explants of gerbera. D. Endogenous slow-growth bacteria in shoot tip culture. E. In vitro pro-
liferation of shoots of gerbera. F. Multiple shoot induction in gerbera. G. Isolated shoots from stage of multiplication. H. In vitro rooting of gerbera plantlets. I. Individual plantlet
rooted in vitro. J. Acclimatized plantlets of gerbera in substrate containing coconut fiber and vermiculite. K. Adult plants of micropropagated gerbera flowering after four months
of cultivation in greenhouse conditions. L—Morphological variations in plantlets from clusters of gerbera cv. ‘AL101’ multiplied in MS medium with 1.0 mg L−1 of BAP, Bar = 1.0 cm,
LH—hyperhydricity in leaves. M. chlorophyll mutation plant from long-term micropropagation culture of gerbera.

with 2 mg L−1 (‘Tobia’) or 3 mg L−1 BA (‘Arianna’ and ‘Bonnie’) (Son
et al., 2011). The potential to induce callus and regenerate shoots is also
dependent on the genotype (Jerzy and Lubomski, 1991; Orlikowska et
al., 1999; Reynoird et al., 1993). Jerzy and Lubomski (1991) showed a
useful protocol for direct and indirect regeneration of shoots of 28 cul-
tivars of gerbera from petiole explants on MS medium supplemented
with 3.0 mg L−1 BA and 0.5 mg L−1 IAA. Orlikowska et al. (1999) ob-
served differences in the regeneration potential of petiole explants of
‘Mariola’ (94.1%), ‘Boy’ (55.3%) and ‘Rebecca’ (47.5%), while the maxi-
mum mean number of shoots/explant was obtained in ‘Mariola’ (4.9),
followed by ‘Boy’ (2.5) and ‘Rebecca’ (2.3). Reynoird et al. (1993) ob-
served high differences in young leaves (≤2 mm) that regenerated
shoots between two clones, clone 10 (83.8–97.8%) and clone 11
(36.6–49.4%), both from the same cross between G. jamesonii and
G. viridifolia, relative to two wild species G. viridifolia (50–65.6%) and
G. piloselloides (7.1–39.7%). Topoonyanont and Dillen (1988) observed
that shoot induction from capitulum tissue was observed only in
cultivars with orange, yellow or red flowers, but no regeneration was
possible from cultivars with pink flowers, suggesting a relation between
flower color and organogenic capacity in gerbera. Shabanpour et al.
(2011) also observed that orange and pink gerbera cultivars showed
differences in shoot regeneration potential (88 and 44%, respectively)
and number of shoots/explant (3.1 and 1.7, respectively) on MS medi-
um with 4 mg L−1 BA and 0.1 mg L−1 IAA using capitulum explants.
These last two studies suggest some level of recalcitrance in shoot or-
ganogenesis in cultivars with pink flower color. The percentage of callus
induction (5–43%), shoot regeneration (57–95%) and number of shoots/
explant (1.1–5.1) were all genotype-dependent in 10 genotypes of ger-
bera obtained from tTCL culture of flower buds (Nhut et al., 2007). Misra
et al. (2010) observed differences in shoot regeneration capacity when
using MS culture medium with 0.5 mg L−1 BA, 0.5 mg L−1 TDZ and
1.0 mg L−1 IAA, being able to rank 7 cultivars based on their regenera-
tion ability: ‘Dana Ellen’ (11.2 shoots/explant) > ‘Salvador’ (9.8) >
‘Rosaline’ (8.6) = ‘Silvester’ (8.0) > ‘Sunway’ (7.0) > ‘Goliath’ (5.0) =

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j.biotechadv.2013.05.008
6 J.C. Cardoso, J.A. Teixeira da Silva / Biotechnology Advances xxx (2013) xxx–xxx
‘Zingaro’ (5.4) for peduncle culture, and ‘Salvador’ (10.5 shoots/
explant) > ‘Dana Ellen’ (8.8) = ‘Goliath’ (8.0) > ‘Zingaro’ (7.2) >
‘Rosaline’ (6.0) > ‘Silvester’ (5.2) = ‘Sunway’ (5.0) for capitulum
organogenesis.
2.1.4.2. Physiological condition of the mother plant. Flower bud age and
position of the explants influences gerbera in vitro organogenesis.
Young flower buds (7-d) stimulated callus production while 10- and
14-d flower buds stimulated direct shoot organogenesis on the
same medium; the highest survival (98%), direct shoot induction
and number of shoots/explant were observed from 10-d-old flower
buds in tTCL culture (Nhut et al., 2007). Similar results were observed
in leaf organogenesis in two progenies from G. jamesonii × viridifolia,
and from wild types G. viridifolia and G. piloselloides (Reynoird et al.,
1993). These authors observed that very young leaves (≤2 mm)
were best for shoot regeneration and that the use of mature leaves
(≥6 mm) did not result in shoot regeneration in four genotypes test-
ed. Callus induction and indirect organogenesis from bracts was
higher from the fully developed capitulum than from 1 or 2-d old
(young) flower buds, but more direct shoots formed mainly from
the axils of bracts from these young flower buds than in fully devel-
oped inflorescences (Schum and Busold, 1985). Pierik et al. (1975)
noted that fully developed inflorescences induced shoots better
than young inflorescences, although portions of immature inflores-
cences between 0.5 and 0.7 cm in diameter showed better shoot re-
generation and number of shoots/explant than from developed
inflorescences (Laliberte et al., 1985). Son (2007) also observed that
bud flowers showed a lower contamination rate and explant death,
best survival percentage and number of shoots/explant than other
kinds of tissues such as shoot tip, capitulum, leaf segments and
peduncles.
Pierik and Segers (1973) observed a higher number of shoots/
explant from inflorescences collected in June and July, relative to
other months, probably because of high temperatures during this
period of the year. The same authors described that inflorescences
could be cut into 4 or 6 equal parts to give best shoot regeneration
from this type of explant. In the culture of G. jamesonii ovules, a
high percentage of callus was obtained in spring for cv. ‘24-2’
(30–35%) and ‘23-6’ (20–25%), and in Autumn for cv. ‘86122’ (10–15%)
and ‘88274’ (20–25%), but callus production was not correlated with
shoot regeneration, and highest morphogenesis was observed in autumn
(Tosca et al., 1999).
2.1.4.3. Sanitation conditions of mother plant and explant sterilization. In
general, most studies present a method to sterilize explants, but they
failed to describe the conditions and treatments in which mother
plants are grown. The absence of this information increases the difficul-
ty in repeating the same conditions and, consequently, the same results
are difficult to obtain, mainly related to explant establishment. Explants
from mother plants cultivated in a greenhouse in pot-culture with coco-
nut fiber, drip local irrigation, fertigation (semi-hydroponic culture)
and insecticidal/fungicidal spraying according to horticultural stan-
dards can result in better results for the growth of gerbera donor plants
in micropropagation than plants grown in soil. Also, based on five-years'
experience of research with gerbera micropropagation in a commercial
lab (Cardoso, personal communication), explants from young shoots
with 7–10-days-old (Fig. 1A) or young inflorescences tend to be less
infected than those derived from older tissues and provide an efficient
way to establish gerbera in vitro using shoot tip culture (Cardoso, per-
sonal observation).
Different methods, products and times of explant exposure tend to
depend on the type and origin of explants. In general, most experi-
ments using different types of explants, cultivars (Tables 1, 2) and
originating from different regions of the world used commercial
products based on chlorine or mercury as the main active com-
pounds. Although most of these experiments indicated the method
Please cite this article as: Cardoso JC, Teixeira da Silva JA, Gerbera micropropagation, Biotechnology Advances (2013), http://dx.doi.org/10.1016/
j.biotechadv.2013.05.008
for sterilization, few papers made an effort to inform on the efficiency
of the method, making it difficult to compare the efficiency of each
methodology or product used.
A 0.1% carbendazim solution for 5 min was a useful fungicidal
pre-treatment followed by surface sterilization with 0.1% of HgCl2 for
7 min to establish flower bud explants (Son et al., 2011). Shabanpour
et al. (2011) washed capitulum explants with tap water for 20 min
then soaked them in a 1.5% (w/v) solution of Ca(ClO)2 for 10 min,
followed by 0.1% (w/v) of mercuric chloride (HgCl2) before rinsing
three times in sterile distilled water (SDW). In another protocol, also
with capitulum explants, Rezende et al. (2008) used 2% (v/v) NaOCl
for 60 min to sterilize explants, although the authors failed to describe
the percentage survival and explant contamination. Young leaves and
capitula were rinsed in running tap water and surface sterilized in
0.1% (w/v) HgCl2 solution for 3 min, followed by a wash in SDW for
5 min (Tyagi and Kothari, 2004). A similar protocol was used by
Paduchury et al. (2010) to sterilize young leaves (4–6 mm) and by
Misra et al. (2010) to sterilize young capitulum explants. Cardoso and
Teixeira da Silva (2012) used 70% alcohol for 2 min, then 1% (v/v) for
10 min, NaOCl for 20 min and finally a rinse three times in autoclaved
distilled water and obtained 20–30% of contamination-free explants
from shoot tip culture. Naz et al. (2012) used only NaOCl (20%) for
20 min but not obtained contamination-free explants.
Shabbir et al. (2012) tested the effect of three concentrations of
NaOCl (5, 10 and 15%, representing 6–14% active chlorine) for 6 min
in continuous agitation on cv. ‘Sunglow’ leaf explant necrosis, con-
tamination and survival. Although the minimum fungal (26%) and
bacterial (18%) contamination were obtained with 15% NaOCl, a
high percentage of explant necrosis was observed in this treatment,
and the best results for survival level of explants (26%) was obtained
with 10% NaOCl, with 23 and 47% of explants being contaminated by
bacteria and fungi, respectively. Nhut et al. (2007), washed recepta-
cles 1.5–2.0 cm in diameter as explants of 7 to 14-d old gerbera
flowers (young) in tap water for 20 min, detergent solution for
15 min and washed again in tap water for another 2 h, then rinsed
six times with SDW and treated the explants with 7% (w/v)
Ca(ClO)2 solution for 25 min, followed by six rinses in SDW.
Physical treatments, such as sonication, that apply ultrasound to-
gether with chemical disinfection, are a possible alternative method to
reduce explant contamination in micropropagated species (e.g., of ma-
ture Paphiopedilum explants; Liao et al., 2011). Even though no studies
related to the use of sonication in gerbera micropropagation were
found, this technique could serve as a possible way of reducing microbi-
al contamination and improving explants survival during in vitro
establishment.
2.1.4.4. Culture media and type of explants. The culture medium, to-
gether with the type of tissue or organ used as the explant, is proba-
bly the most studied factor in gerbera organogenesis (Table 1). MS
medium salts with few modifications has shown the best results in
gerbera micropropagation and has been used in most experiments
to date although some authors used N6 (Chu, 1978), B5 (Gamborg
et al., 1968), DKW (Driver and Kuniyuki, 1984), or LS (Linsmaier
and Skoog, 1965) media for culture establishment (Chen et al.,
2006; Mandal and Datta, 2002; Parthasarathy et al., 1996; Verma
and Anand, 2006), as described by Kanwar and Kumar (2008).
Gerbera is an herbaceous and fast-growing annual plant that requires
high quantities of salts in the culture medium to improve micro-
propagation, mainly at the multiplication stage. These characteristics
explain the success of full or ½ MS culture medium used in most ex-
periments to micropropagate this species, because a high salt concen-
tration is included in these formulations relative to other media
(Table 2).
Rezende et al. (2008) obtained regenerated plants of cv. ‘Jaguar
Cream’ from indirect callus organogenesis from capitulum in ½ MS
with 6.0 mg L−1 BA and 0.5 mg L−1 IAA. Organogenesis from capitulum
 J.C. Cardoso, J.A. Teixeira da Silva / Biotechnology Advances xxx (2013) xxx–xxx
explants (Barbosa et al., 1994; Huang et al., 1987; Topoonyanont and
Dillen, 1988) and shoot tip culture (Cardoso and Teixeira da Silva,
2012) of several gerbera cultivars was also observed in ½ MS media.
Shabanpour et al. (2011) showed that MS with half the concentration
of NH4NO3 and KNO3 was the best medium for maximum shoot regener-
ation (86%) and number of shoots/explant (2.6) during shoot direct re-
generation from capitula. Constantinovici and Sandu (1995) observed
that 85 mg L−1 of NaH2PO4 added to MS medium increased the regener-
ation of plantlets from vegetative and reproductive meristems, relative to
full-strength MS. Orlikowska et al. (1999) used the same MS medium
and added 85 mg L−1 NaH2PO4, 20 g L−1 sucrose, 20 mg L−1 ADS,
0.5 mg L−1 BA, and 0.05 mg L−1 IAA to regeneration three gerbera cul-
tivars (‘Mariola’, ‘Boy’ and ‘Rebbeca’) from petiole explants. Posada et
al. (1999) showed that regeneration of shoots from 1-cm floral buds
was possible by using ½ MS macronutrients combined with Heller's mi-
croelements and vitamins (Heller, 1953), described as MS-H medium. In
most of these studies, even though the capitulum was made up of multi-
ple tissues, including bud flowers, receptacles, bracts, peduncles, ray
flowers, trans-flowers, disc flowers, ovules, and others, the exact tissue
used was never reported, nor was the source of shoots ever confirmed
(Table 1), which makes the repeatability of such published protocols vir-
tually impossible. Misra et al. (2010) showed that capitulum explants
were better than peduncle and sepal explants, and obtained more
shoots/explant for ‘Salvador’, ‘Goliath’ and ‘Zingaro’ from capitulum tis-
sue, although peduncle explants were better than other tissues for
‘Dana Ellen’, ‘Rosaline’, ‘Silvester’ and ‘Sunway’, although in the latter
case, shoots from peduncle explants were obtained via an indirect
route via the formation of callus while the capitulum regenerated shoots
directly. Kumar and Kanwar (2006) tested the induction and regenera-
tion of callus from petals and leaves, and observed that shoots could
only be regenerated from callus derived from petal explants in MS medi-
um with 1–2 mg L−1 NAA rather than Kin, BA or 2,4-D at the same con-
centrations. The regeneration of shoots from petal-derived-callus
explants was possible only in MS media with 3% sucrose plus BA and
IAA or BA and NAA, and the best combination of PGRs was 2.0 mg L−1
BA and 0.5 mg L−1 IAA with 53% of all callus producing an average of
5 shoots/explant (Kumar and Kanwar, 2006).
PGRs are needed to induction organogenesis in gerbera. Several
types and concentrations of cytokinins (Ck), auxins (Aux) and Ck:Aux
ratios were tested in gerbera (Table 1); the ratio strongly influenced cal-
lus induction or direct organogenesis of different cultivars and explant
types (Kanwar and Kumar, 2008). BA at 1 to 10 mg L−1 was frequently
used and tested to induce organogenesis in gerbera. In three gerbera cul-
tivars (‘Mariola’, ‘Boy’ and ‘Rebecca’), Orlikowska et al. (1999) observed
that the best shoot regeneration from petiole-derived callus occurred in
media containing BA (2.2 μmol L−1) and IAA (0.3 μmol L−1). Both the
replacement of BA by TDZ (0.2 μmol L−1), with a concomitant increase
in concentration of BA to 4.4 μmol L−1 did not result in a higher percent-
age of shoot regeneration and number of shoots/explant, not supporting
the results obtained by Reynoird et al. (1993), who observed an im-
provement in the regeneration from mature leaves by adding 0.05 or
0.5 μmol L−1 TDZ to the culture medium. However, Reynoird et al.
(1993) used a particular combination of macronutrients, the MS
micronutrients, also supplementing the medium with 100 mg L−1 glu-
tamine, 20 mg L−1 ADS, and 30 g L−1 sucrose for leaf organogenesis of
G. hybrida (crossing between G. jamesonii × G. viridifolia), G. viridifolia
and G. piloselloides, observing that only in clone 10 from G. hybrida did
TDZ promote the regeneration of buds from leaves ≥6 mm (mature
leaves). Rabori and Ghazvini (2009) tested shoot regeneration from ca-
pitulum explants using different TDZ concentrations and obtained the
highest number of shoots on MS medium with 0.1 and 0.5 mg L−1
TDZ with an average of 4.5 shoots/explant. Callus induction and shoot
regeneration from leaf explants was observed in two gerbera cultivars,
‘AV101’ (cut-flower) and ‘AV108’ (pot-culture), and the Ck:Aux ratio
influenced the regeneration and development of gerbera plantlets,
the best percentage of shoot regeneration (83.3%), number of shoots/
Please cite this article as: Cardoso JC, Teixeira da Silva JA, Gerbera micropropagation, Biotechnology Advances (2013), http://dx.doi.org/10.1016/
j.biotechadv.2013.05.008
7
callus clump (9.9) and shoot height (4.9 cm) being observed when
2.0 mg L−1 NAA and 1.0 mg L−1 BA were added to the culture medium
(Aswath and Choudhary, 2002a). Santilla and Rosario (1998) observed
that MS macronutrients combined with Heller micronutrients (Heller,
1953) solid medium (MS-Heller) supplemented with 2.0 mg L−1
BA and 0.1 mg L−1 IAA produced highest number of shoots from
capitulum explants that regenerated into plantlets. Similar medium
was used by Posada et al. (1999) to regenerate shoots from bud flowers,
although a two-step medium was used, starting with 10 mg L−1 BA and
0.1 mg L−1 IAA for 4 weeks, then transfer of explants to reduced BA
(2.0 mg L−1) medium for a further 4 weeks. Mandal and Datta (2002)
obtained good results with MS-Heller medium, but Tyagi and Kothari
(2004), using three types of media, showed lower shoot regeneration
from leaves, capitulum and in vitro shoots in ½ MS + Heller media
than in MS media and in a similar medium described by Murashige et
al. (1974). Basically, Heller's micronutrients differ from MS medium be-
cause of the lower quantities of micronutrients in general, the exclusion
of CoCl2 and MoO4Na (both present in MS) and the addition of AlCl3 and
NiCl2 to the medium (not included in the MS formulation). Tyagi and
Kothari (2004) also observed that the best shoot regeneration in
young leaves (6.8 shoots/explant), young capitulum (10 shoots/explant)
and in vitro shoots (11 shoots/explant) of gerbera yellow cultivar
(no commercial name provided) was obtained when 4 mg L−1 of kine-
tin (Kin) (leaves and in vitro shoot explants) was combined with 0.1 or
0.5 mg L−1 IAA (capitulum explants). Indirect organogenesis and plant-
let regeneration were observed from petioles in MS medium with
0.5 mg L−1 IAA and 5.0 mg L−1 BA, while direct organogenesis was ob-
served in the same medium when the explants were from young inflo-
rescences (Nongmanee and Kanchanapoom, 1995), showing that the
endogenous PGR balance of different explant types is an important fac-
tor in the response of tissues to different concentrations and types of ex-
ogenous PGRs. Hasbullah et al. (2008) showed that petiole (obtained
from in vitro seed germination) organogenesis was dependent on the
type, concentration and different combinations of Ck and Aux. These au-
thors observed highest shoot regeneration (94.3%) and number of
shoots/explant (9.3) on MS medium with 3% sucrose, 2.0 mg L−1 BA
and 0.5 mg L−1 NAA, and also observed that the combination of
2.0 mg L−1 BA with 0.5 mg L−1 IAA or IBA significantly reduced the
percentage of shoot regeneration from petioles (33.6% and 39.7%, re-
spectively) and the number of shoots/explant (3.5 and 5.1, respectively).
Among the Cks, 2.0 mg L−1 of zeatin (Zea) combined with 0.5 mg L−1
IBA showed better shoot regeneration from petioles (83.7%) and num-
ber of shoots/explant (7.4) than when Zea was substituted by Kin
(66.7% and 6.2 shoots/explant), BA (39.7% and 5.1 shoots/explant) and
2-isopentenyladenine (11.9 and 2.9 shoots/explant) (Hasbullah et al.,
2008).
Other components added to the medium such as amino acids and
complex substances can influence gerbera organogenesis. Best survival
rate, regeneration and number of shoots/explant from tTCLs were ob-
served in MS medium with 0.02 mg L−1 TDZ, 0.8 mg L−1 ADS and
10% (v/v) CW (Nhut et al., 2007). These authors observed that the addi-
tion of ADS and CW reduced callus formation but improved direct shoot
regeneration of gerbera in this system, and concluded that it was mainly
because these components favored or activated the endogenous Ck in
plant tissues. Soczek and Hempel (1988) showed that gerbera multipli-
cation rate was not influenced by thiamine, pyridoxine, meso-inositol,
tyrosine or ADS, although nicotinic acid was needed in one of the
three cultivars ‘Clementine’, ‘Saskia’ and ‘Terravisa’ tested. Gantait et
al. (2011) observed a high number of shoots/explant in ‘Sciella’ when
shoot tip explants were cultured in MS medium supplemented with
2.0 mg L−1 BA and 60 mg L−1 ADS.
2.1.4.5. Incubation conditions and physical factors. In general, incuba-
tion conditions like temperature and light intensity/photoperiod are
some of the least studied factors in plant micropropagation, and this
is no different for gerbera. In most gerbera micropropagation studies,
8 J.C. Cardoso, J.A. Teixeira da Silva / Biotechnology Advances xxx (2013) xxx–xxx

Table 3
Main experimental purposes, culture medium and multiple shoot induction in multiplication stage of different gerbera cultivars.

Cultivar Experimental purpose Culture medium Number multiple shoots Reference

AL101 Sterilization methods MS (3% SCR) + 1.0 mg L−1 BA 5.6–6.4 Cardoso and Teixeira da Silva (2012)
Cross II PGRs MS (3% SCR) + 3.0 mg L−1 Kin + 0.5 mg L−1 BA + 0.5 mg L−1 IBA 22.2 Nhut et al. (2007)
Cabana Genetic fidelity MS (4.5% SCR) + 1 mg L−1 BA + 0.1 mg L−1 IAA NP Bhatia et al. (2009)
Sciella PGRs MS + 2.0 mg L−1 BA + 60 mg L−1 of ADS 14.0 Gantait et al. (2010)
Rui Kou Light quality (CCFL) MS (3% SCR) + 1.0 mg L−1 BA + 0.1 mg L−1 IBA NP Wang et al. (2011)
Arianna and Bonie PGRs MS (3% SCR ?) + 3 mg L−1 BA + 0.1 mg L−1 NAA 12.6 and 15.5 Son et al. (2011)
Tobia PGRs MS (3% SCR ?) + 2 mg L−1 BA + 0.1 mg L−1 NAA 11.3 Son et al. (2011)
Not Described PGRs MS (3% SCR) + 10.0 mg L−1 BA 9.0 Naz et al. (2012)

ADS—adenine sulphate; BA—6-benzyladenine; CCFL—cold cathode fluorescent lamps; IAA—indole-3-acetic acid; IBA -indole-3-butyric acid; Kin—kinetine; MS—Murashige and
Skoog (1962) culture medium; NAA—naphtaleneaceticacid; NP—not presented by authors; PGR—plant growth regulator; SCR—sucrose.

these conditions remain fixed (Table 5). The temperature used for all
stages of gerbera micropropagation tends to range from 25 to 27 °C
while the most commonly used photoperiod of studies reported in
this review is 16 h. Only Nhut et al. (2007) used a 12-h photoperiod
for organogenesis of tTCLs explants, obtaining similar results of per-
centage of regenerated explants and number of shoots/explant as
other papers that used a 16-h photoperiod.
Wang et al. (2011) tested different sources of light in the growth
of in vitro ‘Ruikou’ gerbera plantlets, and obtained significant results
with cold cathode fluorescent lamps (CCLF). These authors showed
that CCFL lamps (70% red + 30% blue) resulted in highest number
of leaves (8), leaf length (1.6 cm) and width (1.5 cm), shoot fresh
weight (4.02 g) and SPAD value (9.95) than other combinations of
CCFL color lamps and plant growth fluorescent lamps (regular
lamps used in tissue culture laboratories). However, the highest num-
ber of roots (6.4), root length (2.86 cm), total and root fresh weight
(7.44 and 4.06 g), and total, root and shoot dry weight (6.18, 2.94
and 3.24 g) were obtained with 100% white CCFL.
2.2. Multiplication stage
In general, the number of shoots/explant is relatively high (>5), but
it is genotype-dependent (Table 3). In a commercial laboratory, in
which more than 50 gerbera cultivars were used, JC Cardoso observed
that shoot multiplication rate can vary anything from 1 to 10/explant.
However, a higher number of multiple-shoot induction was obtained
by Gantait et al. (2010) with ‘Sciella’ (14 shoots/explant) and by proge-
nies from cross II (22.2 shoots/explant) obtained by Nhut et al. (2007).
However, some cultivars are very recalcitrant to multiple shoot induc-
tion. Interestingly, the number of shoots formed is related to inflores-
cence color and some cultivars with red, orange, yellow or pink
flowers that have very wide petioles showed low propagation of multi-
ple shoots in the same media in which other cultivars produced a high
number of shoots/explant (Cardoso et al., unpublished results)
(Fig. 1E and F). Topoonyanont and Dillen (1988) could induce multiple
shoots of orange, red and yellow cultivars, but not of a pink cultivar,
even when a high concentration of Kin (25 mg L−1) was used.
2.2.1. Factors that affect shoot multiplication
Several factors affect shoot multiplication of gerberas and are used
in commercial laboratories to control and produce millions of plant-
lets of several cultivars of gerbera in a short period of time. The
main factors that influence the multiplication stage of gerbera are: ge-
notype, culture medium, growth conditions and the propagation
method (Table 3).
2.2.1.1. Genotype. Harel et al. (1993) observed different responses in
the multiplication rate among 10 cultivars, and classified them as
high-multiple-shoot-induction (8) cultivars (‘Ansofie’, ‘Terracerise’
and ‘Lablinel’), moderate (6–7) (‘Maria’, ‘Shangha’ and ‘Fresultane’)
and low-multiple-shoot-induction cultivars (4–5) (‘Raisa’, ‘Fredigor’,
‘Terramaxima’ and ‘Fredibel’). Son et al. (2011) also observed that
days needed to initiate shoot induction, media requirements and num-
ber of shoots required for multiple shoot induction are genotype-
dependent in three gerbera cultivars. They observed that ‘Tobia’ needs
an average of 9.6 days for shoot initiation with an average of 11.3 mul-
tiple shoots/inoculated shoot induced on MS medium supplemented
with 2 mg L−1 BA and 0.1 mg L−1 NAA, while ‘Arianna’ and ‘Bonnie’
were best propagated on MS containing 3 mg L−1 BA and 0.1 mg L−1
NAA, and needed 6.6 and 5.4 days to initiate shoots, forming 12.6 and
15.5 shoots/culture, respectively. Topoonyanont and Dillen (1988)
also observed that differences in multiplication rate depended on the
cultivar tested and on the Ck concentration used. A cultivar with orange
inflorescences (described by the authors as cv. ‘Orange’) showed best
multiplication rate with 5 or 7.5 mg L−1, yellow with 10–15 mg L−1
and red with 25 mg L−1, when Kin served as the Ck.
2.2.1.2. Culture medium. The most commonly used medium in the ger-
bera multiplication stage is full MS medium and the type and concen-
trations of PGRs has been extensively studied (Table 3). Sousa et al.

Table 4
Main experimental purposes, culture media, and rooting percentage of individualized shoots in different gerbera cultivars.

Cultivar Experimental purposes Culture medium Rooting Percentage Reference

Jaguar Cream PGRs 1/2 MS (3% SCR) + 0.1% AC + 4.0 mg L−1 NAA NP Rezende et al. (2008)
Arianna, Bonnie, Tobia PGRs MS + 2.0 mg L−1 NAA 86%, 96%, 100% Son et al. (2011)
Sunglow PGRs MS (3% SCR) + 1.5 mg L−1 IAA 97.7% Shabbir et al. (2012)
Not described 1/2 MS (3% SCR) (without PGRs) 100% Chakabrarty and Datta (2008)
AL101 1/2 MS (3.5% SCR) + 0.05 mg L−1 IBA 100% Cardoso and Teixeira da Silva (2012)
Cross II PGRs 1/2 MS (3% SCR) + 1.0 mg L−1 IBA NS Nhut et al. (2007)
Cabana Genetic fidelity 1/2 MS (4.5% SCR) + 1.0 mg L−1 IBA 100% Bhatia et al. (2009)
Sciella PGRs MS + 0.5 mg L−1 IAA
Not described PGRs MS (3% SCR) + 2 mg L−1 NAA 60.5% Hasbullah et al. (2008)
Red colour cut-flower gerbera Ex vitro rooting + quickly dipping in 2000 mg L−1 IBA 50–60% Kumar and Kanwar (2006)
Rui Kou Light quality (CCFL) 1/2 MS (3% SCR) + 0.5 mg L−1 IBA + 0.5 mg L−1 IAA NP Wang et al. (2011)
Not described PGRs MS (3% SCR) + 10.0 mg L−1 NAA 80% Naz et al. (2012)

CCFL—cold cathode fluorescent lamps; IAA—indole-3-acetic acid; IBA -indole-3-butyric acid; MS—Murashige and Skoog (1962) culture medium; NAA—naphtaleneaceticacid;
NP—not presented by authors; PGR—plant growth regulator; SCR—sucrose.

Please cite this article as: Cardoso JC, Teixeira da Silva JA, Gerbera micropropagation, Biotechnology Advances (2013), http://dx.doi.org/10.1016/
j.biotechadv.2013.05.008
 J.C. Cardoso, J.A. Teixeira da Silva / Biotechnology Advances xxx (2013) xxx–xxx
Table 5
Growth conditions in all stages of micropropagation experiments of different cultivars of gerbera.
Cultivar Establishment and growth conditions
Orange and Pink 25 ± 2 °C, 30-d darkness, 67.5 μmol m−2 s−1, 16-h Phot
Jaguar Cream 25 ± 2 °C, 30-d darkness, 43 μmol m−2 s−1, 16-h Phot
Arianna, Bonnie, Tobia 25 ± 2 °C, 97.2 μmol m−2 s−1, 16-h Phot
Sunglow 30-d darkness, 25 ± 1 °C, 27 μmol m−2 s−1, 16-h Phot
Cabana 25 ± 1 °C, 47 μmol m−2 s−1, 16-h Phot
Non-described 25 °C, 36 μmol m−2 s−1, 16-h Phot
AL101 dark 14-d, 26 ± 1 °C, 30 μmol m−2 s−1, 16-h Phot
Jaimy and Mammut 30-d dark at 21 ± 2 °C and 25 ± 2 °C, 40 μmol m−2 s−1,
16-h Phot
Ten Interspecific crosses 25 °C, 40.5 μmol m−2 s−1, 12-h Phot
Sciella (Yellow flowers) 25° ± 2 °C, 30 μmol m−2 s−1 16-h Phot
Non-described NI
Salvador, Goliath and Zingaro 25 ± 2 °C, 60 μmol m−2 s−1, 16-h Phot
Dana Ellen, Rosaline, 25 ± 2 °C, 60 μmol m−2 s−1, 16-h Phot
Silvester, Sunway
Red colour cut flower gerbera 25 ± 2 °C
Not-described 22 ± 2 °C, 33–40 μmol m−2 s−1, 16-h Phot
Rui Kou (CCFL lamps) 24 ± 1 °C, 36 μmol m−2 s−1 (70% Red + 30% Blue),
12-h Phot
CCFL—cold cathode fluorescent lamps; equal EGC—equal establishment and growth conditions; NI—not indicated by authors; Phot—photoperiod; similar EGC—establishment and
growth conditions without a dark period.
(2006) showed that media containing 75 and 100% of MS salts was
best for multiplication, and obtained a high multiple shoot induction
(near 12) using a combination of 0.5 mg L−1 IBA and 0.5 mg L−1 BA.
Aswath et al. (2003) observed that ½ MS resulted in faster shoot mul-
tiplication while full-strength MS culture medium resulted in a higher
number of shoots, longer shoots and greater shoot weight. These au-
thors also observed that all three varieties (GJ-1, GJ-2 and GJ-3)
showed highest shoot number/explant in full-strength MS medium
supplemented with 1.0 mg L−1 BA or 5.0 mg L−1 Kn, both combined
with 0.1–0.2 mg L–1 IAA. Budi (2000) obtained most multiple shoots in
full-strength MS medium with 500 mg L−1 casein hydrolysate,
1.0 mg L−1 BA and 0.2 mg L−1 NAA. Son et al. (2011) obtained a better
shoot multiplication rate in MS with 3.0 mg L−1 BA and 0.1 mg L−1
NAA, ranging between 5.8 and 15.5 in four gerbera cultivars. A combina-
tion of 2.0 mg L−1 BA and 0.3 mg L−1 NAA resulted in the most shoots/
explant when MS medium was used in the multiplication stage (Feng et
al., 2009). Posada et al. (1999) showed best multiple shoot induction
with 1 or 2 mg L−1 BA in MS-H medium. Parthasarathy and Nagaraju
(1999) observed that in ‘SWM’ and ‘Dilmaya’ more multiple shoots
formed when shoot explants were propagated in vitro in MS medium
with 0.5–1.0 mg L−1 BA. Normally, higher concentrations of BA (more
than 2.0 or 3.0 mg L−1) result in reduction of shoot multiplication
and increase symptoms of hyperhydricity, but some authors, such as
Naz et al. (2012), obtained best shoot multiplication using 10 mg L−1
BA (9 shoots/explant) without reporting any hyperhydricity.
Bouman et al. (2001) showed that MS was superior to DKW medi-
um in shoot multiplication. Cks such as Kin and TDZ can be used in
the multiplication stage of gerbera micropropagation. Sahavacharin
(1985) could multiply gerbera most rapidly on MS medium with
0.75 mg L−1 IAA and 12 mg L−1 Kin. Tyagi and Kothari (2004) ob-
served that 2 mg L−1 Kin combined with 0.5 mg L−1 phenylacetic
acid (PAA) promoted best multiplication rate and better recovery of
shoots than 0.5 mg L−1 IAA. Reynoird et al. (1993) used 0.25 mg L−1
BA combined with 0.25 mg L−1 Kin and 0.45 mg L−1 IAA for the multi-
plication stage of G. hybrida (G. jamesonii × G. viridifolia).
In conventional micropropagation, the use of a carbon source is
necessary because of the low photosynthetically photon flux density
(PPFD) and in vitro low CO2 concentrations (Kozai et al., 1997; Xiao
et al., 2011). The most common carbon source in micropropagation
is sucrose and, in gerbera, the best concentration for all stages of
micropropagation varies between 2 and 3% (Kanwar and Kumar,
2008). Huang and Chu (1985) found the best sucrose concentration
to be 2% for multiple-shoot induction (3 shoots/explant) in MS
Please cite this article as: Cardoso JC, Teixeira da Silva JA, Gerbera micropropagation, Biotechnology Advances (2013), http://dx.doi.org/10.1016/
j.biotechadv.2013.05.008
9
Multiplication Rooting Reference
Shabanpour et al. (2011)
Similar EGC Rezende et al. (2008)
Equal EGC Son et al. (2011)
Similar EGC Shabbir et al. (2012)
Equal EGC Bhatia et al. (2009)
Equal EGC Chakabrarty and Datta (2008)
Similar EGC Similar EGC Cardoso and Teixeira da Silva (2012)
Rabori and Ghazvini (2009)
Equal EGC Nhut et al. (2007)
Equal EGC Gantait et al. (2010)
NI Hasbullah et al. (2008)
Misra et al. (2010)
Misra et al. (2010)
Kumar and Kanwar (2006)
Equal EGC Equal EGC Naz et al. (2012)
Equal EGC Wang et al. (2011)
medium with half the concentration of macro-nutrients, 5.0 mg L−1
BA and 0.1 mg L−1 IAA, solidified with 10 g L−1 Bacto-agar. In that
study, the absence of sucrose resulted in a poor multiplication rate
(b1%) while 1, 4 and 8% sucrose resulted in similar multiple-shoot in-
duction i.e., 2%.
Shailaja (2002) and Jerzy and Lubomski (1991) obtained the
highest number of shoots/explant with 3 mg L−1 BA. Chakabrarty
and Datta (2008) also observed that BA was superior for shoot multi-
plication than other Cks, such as Kin and TDZ, and showed that the best
multiplication rate (17:1) took place in 8.8 μmol L−1 (2.0 mg L−1) BA.
However, Nhut et al. (2007) observed that the multiplication rate was
best (22.2:1) in ½ MS containing 0.5 mg L−1 IBA), 0.5 mg L−1 BA,
and 3.0 mg L−1 Kin.
The best pH of the culture medium for shoot proliferation varies
between 5.7 and 6.7 (Aswath and Choudhary, 2002b).
Another factor that strongly affects the multiplication of gerbera and
is directly correlated with the media components such as nutrients,
PGRs and sucrose is the method used to sterilize the media. Recently,
Cardoso and Teixeira da Silva (2012) observed that the chemical steril-
ization of media with chlorine dioxide (cold sterilization) resulted in a
small increase in multiple shoot induction and better quality of plantlets
of gerbera cv. ‘AL101’ than that obtained in autoclaved media (high
temperature sterilization). Pan and Van Staden (1999) reported that
autoclaving led to the degradation of culture media components,
changing its composition.
2.2.1.3. Growth conditions. Physical growth conditions are one of the
factors for which there is a lack of detailed information in gerbera
micropropagation, most authors using similar temperature, light in-
tensity and photoperiod in all stages of micropropagation (Table 5).
Gerbera multiple-shoot induction increased as PPFD increased from
800 (2.83 shoots/explant) to 3000 lx (3.5 shoots/explant) using fluo-
rescent lamps under a 16-h photoperiod and at 27 ± 2 °C (Huang
and Chu, 1985).
The type of tissue culture container is another factor that influ-
ences growth in tissue culture (e.g. hybrid Cymbidium; Teixeira da
Silva and Tanaka, 2009). Internal gaseous composition is one of the
factors that affects the choice of container used and which directly af-
fects the development of plants in vitro, due to ethylene production
and build-up. Woltering (1990) showed that propylene and butane
gases were found in polypropylene and polyvinylchloride, but not in
glass containers. They also showed that an additional unidentified
gas, apparently phytotoxic to gerbera micropropagation, was found
10 J.C. Cardoso, J.A. Teixeira da Silva / Biotechnology Advances xxx (2013) xxx–xxx
only in polypropylene containers, causing a substantial loss in the
quality of propagated plantlets.
2.3. Rooting and elongation stage
Shoot from multiplication stage are individualized (Fig. 1G) and
transplanted for rooting and elongation culture medium. The rooting
and elongation stages can be combined into one stage in gerbera
(Fig. 1H and I), while rooting can be induced in vitro (Cardoso
and Teixeira da Silva, 2012) or in vivo (Huang and Chu, 1985;
Podwyszynska and Gabryszewska, 2003). Constantinovici and Sandu
(1995) observed that the rooting of gerbera is easy and possible in
media without PGRs, and concluded that plants that rooted in vitro
showed better acclimatization than unrooted ones. Most experiments
tested the effects of genotype and culture media (principally the type
and concentration of Aux) (Table 4). In general, normal micro-
propagated gerbera plantlets are easy to root and high rooting per-
centages (80–100%) are frequently observed (Fig. 1H, Table 4). The
time required for rooting and elongation of gerbera is around 30
days after the inoculation of shoots (Aswath and Choudhary, 2002a;
Cardoso and Teixeira da Silva, 2012).
2.3.1. Factors that affect rooting of gerbera
2.3.1.1. Genotype and culture medium. Cardoso and Teixeira da Silva
(2012) used ½ MS salts with 35 g L−1 sucrose and a very low concentra-
tion of IBA (0.05 mg L−1) to root and elongate gerbera shoots. Rooting
gerbera shoots is possible on PGR-free medium (Constantinovici and
Sandu, 1995; Posada et al., 1999), but in general, rapid rooting, best
rooting percentage and highest number of adventitious roots/shoot are
obtained when some type of Aux was added to the medium, such as
IAA (Nongmanee and Kanchanapoom, 1995; Sahavacharin, 1985),
NAA (Pierik and Sprenkels, 1984; Rezende et al., 2008) or IBA
(Cardoso and Teixeira da Silva, 2012). Sahavacharin (1985) observed
that 0.5 mg L−1 IAA added to MS media best induced adventitious
roots in gerbera. Shabanpour et al. (2011) showed that 100% of two cul-
tivars (‘Pink’ and ‘Orange’) could be successfully rooted in MS media
with IAA or NAA, but most roots (4.6–5.1 roots/shoot) could be obtained
with 3 mg L−1 IAA. Shabbir et al. (2012), testing different concentra-
tions of NAA and IAA for rooting of ‘Sunglow’ shoots, obtained best
rooting percentage (97.7%), number of roots/shoot (7.6) and root length
(7.33 cm) in MS medium with 1.5 mg L−1 IAA. NAA (0.5–2.0 mg L−1)
resulted in lower rooting percentage (36.7–60.7%) than IAA, indepen-
dent of the concentration. In contrast, Pierik and Sprenkels (1984) and
Murashige et al. (1974) obtained 100% rooting in shoots cultured in
MS medium containing 1 or 3 mg L−1 NAA, better than IAA for ‘Fleur’
and ‘Florence’ and the choice of genotype affected the rooting response
to different types of Aux. Shailaja (2002) also observed 100% rooting
on MS with 2 mg L−1 NAA. The media used in stages prior to rooting,
such as shoot multiplication media, tend to affect the subsequent
rooting stage. In ‘Jaguar Cream’, an increasing concentration of NAA
(0.0–4.0 mg L−1) in ½ MS also showed an increase in the number
(1.6–5.0) and length (3.4–5.0 cm) of roots (Rezende et al., 2008). Feng
et al. (2009) observed that ½ MS medium was best for rooting gerbera
stem segments when 0.2 mg L−1 NAA was added. Son et al. (2011)
obtained the best rooting percentage (86, 96 and 100%), days taken
for rooting to start (16.2, 15.2 and 17.6) and number of roots/shoot
(3.8, 9.4 and 7.2) in MS medium with 2.0 mg L−1 NAA in three cultivars
(‘Arianna’, ‘Bonnie’ and ‘Tobia’). Naz et al. (2012) showed that best
rooting percentage (80%) was obtained using 10.0 mg L−1 NAA. IBA
resulted in 100% adventitious root induction in ‘AL101’ (Cardoso and
Teixeira da Silva, 2012) in which good quality plantlets and roots were
obtained with a low concentration of IBA (0.05 mg L−1), main when
the culture media was chemically sterilized with ClO2 rather than
autoclaved media, suggesting that autoclaving can altered the composi-
tion of the media, mainly the nutrients and PGRs. The use of Cks in the
Please cite this article as: Cardoso JC, Teixeira da Silva JA, Gerbera micropropagation, Biotechnology Advances (2013), http://dx.doi.org/10.1016/
j.biotechadv.2013.05.008
rooting media, such as 0.5 mg L−1 BA, can reduce the percentage of
rooting in gerbera (Sousa et al., 2006).
The mineral composition of the medium influences rooting in ger-
bera (Table 4). Sousa et al. (2006) observed that when MS medium
was reduced to 25% (80% rooting) of its original concentration (near
to 40% rooting), the percentage rooting of ‘Ornela’ increased. Posada
et al. (1999) also observed improved rooting percentage in gerbera
shoots when MS salt concentration was reduction, obtaining best re-
sults with ½ MS-H medium without IAA. Some authors did not ob-
serve an effect of mineral concentration of MS medium on the
rooting percentage of ‘Appelbloesem’ (Barbosa et al., 1992, 1993),
suggesting the genotype dependence of the rooting response and nu-
trient requirements for this growth stage (Table 4).
2.3.1.2. System used for rooting. Rooting and elongation are the easiest
stages of gerbera micropropagation because of the high and rapid re-
sponses of most gerbera cultivars to rooting (Table 4). In general, is
possible to obtain 100% rooting induction within 30-d after the
inoculation of isolated shoots (Cardoso and Teixeira da Silva, 2012).
However, this stage probably represents the highest costs to
micropropagation because considerable lab space and media are re-
quired while much labor is required for medium preparation and to
isolate single shoots. The rooting stage is labor intensive and expen-
sive (IAEA-TECDOC, 2004), but uncertain data about the costs of this
stage of micropropagation have been published.
Wang et al. (2008) tested new alternatives to substitute the high
costs of agar as substrate for gerbera rooting in MS medium, and
showed that high rooting percentage (96.6%), plant quality and survival
rate of transplants (97.6%) was possible when plantlets were rooted in
grit, although grit was not a successful substrate for rooting, possibly be-
cause they used full-strength MS medium with a high concentration of
BA and a high Ck:Ax ratio (15 mg L−1 BA and 0.2 mg L−1 IBA).
Podwyszynska and Gabryszewska (2003) tested ex vitro rooting of
‘Rebbeca’ in rockwool (Grodan SBS) using filtered red light treat-
ments (3 μmol m−2 s−1) during the night period in greenhouse con-
ditions, observing an increase in rooting percentage from 67.1%
(control) to 98.6% (red light treatment) in winter.
Ruffoni et al. (1992) observed that initial roots were visible 21-d
after in vitro cultivation of shoots and early rooting was obtained in
20% of shoots that rooted ex vitro when a mix of perlite-peat (1:1)
substrate was used.
Another strategy used at the stage of rooting of shoots in
micropropagation is long-term storage at low temperature. The stor-
age of gerbera at low temperatures can provide a solution for the
rapid acclimatization of rooted plantlets to meet seasonal market de-
mands. Hempel and Hempel (1987) showed that it was possible to
store different cultivars of in vitro rooted gerbera at 4 °C for up to
3 months in darkness and up to 6 months in light without affecting
the survival of plants after greenhouse acclimatization.
2.4. Acclimatization
This is one of the most critical phases in gerbera micropropagation
(Fig. 1J). This is primarily because of the tall, tender tissues and the
high and rapid water loss by leaves, caused by dysfunctional stomata
(Cardoso et al., unpublished results) in sucrose-containing media and
other anatomical (Rezende et al., 2008), morphological and physio-
logical disorders caused by the micropropagation of plantlets in
sugar-containing media compared to ex vitro plantlets (Pospišilova
et al., 1999). In conventional micropropagation, plantlets are ob-
served to have altered chlorophyll content, net photosynthetic rate,
stomatal conductance, stomatal density, transpiration rate, and leaf
structure such as a thick cuticle and leaf mesophyll that make accli-
matization difficult (Pospišilova et al., 1999; Xiao et al., 2011).
Huylenbroeck and Debergh (1992) observed two periods of stress in
acclimatized gerbera plantlets. The first occurred some days after
 J.C. Cardoso, J.A. Teixeira da Silva / Biotechnology Advances xxx (2013) xxx–xxx
transplanting to rooting culture medium, when the plants had not yet
rooted, and the second one was the period in which plants were
transferred from weaning to greenhouse conditions. Chakabrarty
and Datta (2008) observed an increase in lipid peroxidation and
H2O2 content in gerbera plantlets, mainly in the first 15 days after
in vitro culture (early stages of acclimatization), resulting in oxidative
stress. The latter authors obtained an increase in survival of acclima-
tized plants (almost 70%) when increasing the relative humidity of air
in the chamber to 80–90%, and acclimatization in these conditions for
15–20 days.
Substrates used to acclimatize gerbera vary depending on the
availability of that country's natural resources, and in most cases, a
mineral, organic or a mix thereof are used to acclimatize gerbera. Co-
conut fiber is an organic substrate that has been used in the cultiva-
tion of several species, including gerbera and presents good results
in the acclimatization of gerbera (Cardoso et al., unpublished results)
(Fig. 1J). Paradiso and De Pascale (2008) observed that the addition of
the upper-layer of coconut fiber to perlite resulted in higher leaf area,
more cut stem inflorescences, and higher contents of P and K in leaves
than only perlite in ‘Leader’. Taha et al. (2010) used garden soil, ver-
miculite, black soil and autoclaved garden soil to acclimatize gerbera
plantlets that had previous rooted in vitro. They observed that garden
soil (black:red soil, 2:1) showed best results with 86 ± 0.9% plantlet
survival, followed by vermiculite with 73 ± 1.3% survival. Rooted
plantlets of micropropagated gerbera were successfully acclimatized
(95% survival rate) using plastic pots with coco peat, red soil and
sand (3:1:1) (Aswath and Choudhary, 2002b). Parthasarathy and
Nagaraju (1999) obtained 95–100% success at the acclimatization
stage using polyethylene bags and soil:sand:farmyard manure
(1:1:1) as the organic-mineral substrate in gerbera ‘SWM’ and
‘Dilmaya’. Son (2007) observed a higher percentage of explant surviv-
al in three gerbera cultivars, Tobia (60%), Arianna (66%) and Bonnie
(74%), previously rooted in vitro using only vermiculite up until the
acclimatization stage. In their study, sand and cocopeat mix 1:1 (v/v)
showed the worst result, with a 27% survival of plantlets. In all these
studies, the exact day after transplanting that acclimatization success
was determined was not indicated.
2.5. Photoautotrophic culture
Another possibility for micropropagation of gerbera is photoauto-
trophic culture. This system is characterized by the use of culture
media without sugar and under high photosynthetic photon flux den-
sity (PPFD) and a high CO2 concentration in the growth environment
to promote photoautotrophism in micropropagated plantlets (Xiao
et al., 2011). Photoautotrophic culture also shows an additional ad-
vantage, cost reduction in commercial micropropagation (Xiao and
Kozai, 2004). Increasing CO2 concentration to 900 ppm and PPFD to
95 μmol m−2 s−1 increased the speed of acclimatization of gerbera
and improved the growth and quality of plants obtained (Huylenbroeck
and Debergh, 1992). These authors concluded that plant growth at a
high CO2 level tested could control water loss effectively. Liao et al.
(2007) observed an increase in fresh weight (4.32 g) and dry mass
(10.6%) of shoots 2–3 cm long and with 3–4 leaves that were cultured
under diffusive ventilation combined with flasks with a gas-permeable
membrane as the flask cap. These authors also observed that under
these conditions, shoots could survive in photoautotrophic culture
(free-sugar medium).
The “Culture-Pack”–rockwool system is another alternative to rooting
micropropagated gerbera shoots with the advantages of culture in
non-sterile conditions and in a photoautotrophic system (sugar-free me-
dium). The gerbera plantlets obtained in this system were normal, more
vigorous and significantly larger than a conventional micropropagation
system in agar medium with sugar and in sterile conditions (Nagae et
al., 2003).
Please cite this article as: Cardoso JC, Teixeira da Silva JA, Gerbera micropropagation, Biotechnology Advances (2013), http://dx.doi.org/10.1016/
j.biotechadv.2013.05.008
11
3. Genetic and epigenetic variations in micropropagation of gerbera
Gerbera is considered to be a fast-growing herbaceous ornamental
that needs 90–120 days to start flowering (Fig. 1K), and it is possible
to evaluate the presence of morphological variations in ex vitro green-
house growth conditions from micropropagated plants caused by ge-
netic or epigenetic variations (Cardoso and Teixeira da Silva, 2012).
However, the occurrence of somaclonal variation in gerbera is a prob-
lem for commercial biofactories and the detection and elimination of
this type of shoot or plantlets needs rapid, efficient, and periodic
monitoring, with the aim of preventing the propagation and acclima-
tization of somaclonal variants together with commercial true-to-
type genotypes. However, in gerbera, no genetic or epigenetic modi-
fications have been observed in most cultivars micropropagated
from shoot tip explants (Cardoso and Teixeira da Silva, 2012;
Gantait et al., 2010; Murashige et al., 1974). Reynoird et al. (1993)
did not observe morphological variations in vegetative and reproduc-
tive phases of gerbera plants obtained from micropropagation.
Cardoso and Teixeira da Silva (2012) also did not observe any
kind of visible morphological alterations in all phases of development
(in vitro and in vivo) of gerbera cv ‘AL101’ when they used shoot tips
as the initial explants for micropropagation. What is of interest is that
most papers dealing with gerbera tissue culture or micropropagation,
primarily from capitulum/leaf organogenesis, never described nor
evaluated the regenerated plantlets under greenhouse conditions,
thus it is difficult to identify or characterize possible mutations. In
some in vitro gerbera cultivars, it is possible to observe chlorophyll
mutants in long-term cultures, that can be observed in vitro or ex vitro
in the acclimatization stage (orange-flowering ‘Dawn’; Fig. 1M), but
this is very rare (1 or 2 plants/1.5 million). This observation was made
from over 6 years of observations from over 1.5 million plantlets pro-
duced (Cardoso, personal observation).
Molecular markers can be used for the characterization and protec-
tion of gerbera cultivars, although the large number of species and cul-
tivars limits the application across such a wide belt of variation, often
compounded by the fact that there is little appetite to research such var-
iation in ornamentals relative to, for example, edible crops. Six of 20
random amplified polymorphic DNA (RAPD) markers (Rusinowski
and Domeradzka, 2012) were selected and useful to detect genetic var-
iation among 8 gerbera cultivars, resulting in polymorphic bands vary-
ing from 28.9 to 40% of differentiating bands, depending of the marker
selected. Gong and Deng (2012) selected 53 robust and informative
EST-SSR markers for use in different works with breeding, genetic stud-
ies and detection of genetic fidelity in gerbera. Bhatia et al. (2011) did
not observed genetic variation from 1744 bands evaluated and obtained
from RAPD and ISSR (inter-simple sequence repeats) markers of ger-
bera cv. ‘Cabana’ plantlets obtained from in vitro capitulum explants,
with only 12 of 35 RAPD and 10 of 32 ISSR markers showing well de-
fined bands and good reproducibility, although in an earlier study by
the same group, Bhatia et al. (2009) evaluated the genetic fidelity of
regenerated plantlets from capitulum, shoot tip and leaf explants of
‘Cabana’ and detected one leaf-derived plantlet with loci different
from the donor plant. These authors emphasized that this variation oc-
curred because leaf-derived plantlets were obtained from callus previ-
ously induced from leaves, while capitulum- and shoot tip-derived
plantles were obtained by direct organogenesis, without an intermedi-
ate callus phase, resulting in true-to-type plantlets. Orlikowska et al.
(1999) also observed morphological and physiological variations in
micropropagated ‘Mariola’ plantlets derived from indirect organogene-
sis of petioles, resulting in dwarf plants that did not flower.
Hyperhydricity, which can be observed in gerbera in vitro cultures
(Fig. 1L), is often associated with the relative humidity of the air in
the growth flask, and with the addition of synthetic Cks, such as BA, to
medium (Debergh et al., 1981; Kataeva et al., 1991; Leshem and Sachs,
1985). Kataeva et al. (1991) observed considerable levels (7.5–24.7%)
of hyperhydricity in gerbera plantlets after adding 4.4 μmol L−1 BA
12 J.C. Cardoso, J.A. Teixeira da Silva / Biotechnology Advances xxx (2013) xxx–xxx
(=1.0 mg L−1). Furthermore, these authors observed that in these
hyperhydric plantlets, there was a loss of apical dominance and a loss
in rooting ability. In our studies (Cardoso et al., unpublished results),
we observed a similar finding for concentrations ≥1.0 mg L−1,
depending on the genotype. Those somaclonal variants showed dwarf-
ing, coriaceous and brittle growth and with a reduced diameter of leaf
lamina, all associated with reduced multiplication ability (Fig. 1L). Dif-
ferently, Gantait et al. (2010) achieved exceptionally high rates of mul-
tiplication and obtained 100% normal plantlets of gerbera cv. ‘Sciella’
when cultured on MS medium with 1.0 mg L−1 BA and 60 mg L−1
ADS. Feng et al. (2009) observed that only concentrations higher than
3.0 mg L−1 of BA increased hyperhydricity in gerbera seedlings cultivat-
ed in vitro (cultivar not described), and also observed that a high
concentration of NAA (>0.3 mg L−1) resulted in chlorotic leaves in
micropropagated plants, showing a genotype-dependence of in vitro
hyperhydricity and other abnormalities. In Gerbera aurantiaca, the
highest concentration of BA (2.25 mg L−1) resulted in 20% hyperhydric
shoots (Meyer and Van Staden, 1988).
Other factors studied that can induce epigenetic aberrations in
gerbera are genotype and the type of explant used to initiate micro-
propagation. Topoonyanont et al. (1999) observed morphological
and anatomical differences between ‘Rosabella’ (bushy) and ‘Sunset’
(non-bushy) micropropagated plantlets. In general, the main shoot
explants were most sensitive to bushiness, which was increased by a
high Kin concentration (~2.0 mg L−1) in the medium (Topoonyanont
and Debergh, 2001; Topoonyanont et al., 1999).
There is a possibility of reducing genetic and epigenetic variations
(morphological, anatomical or physiological disorders observed in
micropropagated plantlets that are not or are rarely transmitted to
progeny and which result from long-lasting changes in the expression
of genome information) in gerbera, as in other species (Smulders and
De Klerk, 2011). Gerbera has several genotypes with similar charac-
teristics, namely color of inflorescences, disease resistance, and pro-
ductivity. The choice of genotype that presents low-sensitivity to
epigenetic and genetic variations is a key alternative to the micro-
propagation of true-to-type genotypes. Breeding companies play an
important part in this process, and need to provide information
about these characteristics in the micropropagation of each cultivar
developed. The choice of correct Ck type and concentration, as well
as the use of shoot tips or direct organogenesis from other explants
is another factor that results in high-fidelity gerbera genotypes
(Bhatia et al., 2009; Cardoso and Teixeira da Silva, 2012). Nazneem
(2004) also observed that the culture medium formulation is another
factor that influences hyperhydricity in cv. ‘IIHR’ of gerbera, and
showed that the substitution of MS culture medium by Quoirin and
Lepoivre (1977) medium supplemented with 30 g L−1 sucrose,
3 mg L−1 BA and 0.5 mg L−1 IAA was best to propagate shoot tips
with least hyperhydricity, while MS culture medium with this same
additives showed high levels of shoot hyperhydricity.
Occasionally, flowering mutants (chimeric inflorescences white/rose
flowers, Cardoso, personal observation) can be observed, and in such
cases, molecular markers would be useful to detect such somaclonal var-
iation. Molecular markers could also serve to create a DNA fingerprint of
superior cultivars, allowing for their commercial protection. The use of
cytogenetic analyses can supplement other alternative and complemen-
tary data to detect genetic changes in micropropagated plantlets, as used
by Gantait et al. (2011) to detect autotetraploid plants obtained by treat-
ments with colchicine.
4. Conclusions
Most studies on the micropropagation of gerbera focus on the in-
fluence of culture medium or culture conditions on the establishment
stage. Slow-growing bacterial contamination is one of the greatest
problems facing commercial gerbera micropropagation and more
studies are required to obtain clean, infection-free explants. Genotype
Please cite this article as: Cardoso JC, Teixeira da Silva JA, Gerbera micropropagation, Biotechnology Advances (2013), http://dx.doi.org/10.1016/
j.biotechadv.2013.05.008
dependence of the results is one of the main factors affecting the re-
generation and multiplication of gerbera shoots. Surprisingly, no pub-
lished papers exist showing an efficient protocol that resulted in a
good regeneration and multiplication system for multiple cultivars.
Associated with this, very rare are the studies that tested physical fac-
tors associated with gerbera micropropagation. The gerbera in vitro
rooting stage produces more than 90% of shoots with roots. However,
acclimatization studies are also lacking, and improved substrates and
environmental conditions for acclimatization are required. Genetic
variations in gerbera are very rare, or are rarely reported, and are nor-
mally correlated with adventitious shoots obtained indirectly through
callus, although most gerbera cultivars are sensible to epigenetic
variations when was cultured on media with high concentrations of
cytokinins. Some recently published papers contain incomplete infor-
mation, i.e. genotype used, description of genotype by the color of in-
florescences, origin of explants (Tables 1–4), incomplete information
about the culture media (sucrose concentration, PGRs used, etc.), or
the lack of important data such as number of multiple shoots
(Table 3) or rooting percentage (Table 4) and are not, based on the
experience of JC Cardoso, reproducible, further accentuating the diffi-
culties that currently exist in gerbera micropropagation research.
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