Acta Physiol Plant (2011) 33:15591570

DOI 10.1007/s11738-011-0736-6

REVIEW

Anther culture in pepper (Capsicum annuum L.) in vitro

Teodora Irikova Stanislava Grozeva

Velichka Rodeva

Received: 25 April 2010 / Revised: 21 December 2010 / Accepted: 13 February 2011 / Published online: 4 March 2011
Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2011

Abstract Pepper (Capsicum annuum L.) is an important N Nitsch basal medium (1969)
vegetable crop that can be improved using plant tissue NN Nitsch and Nitsch basal medium (1969)
culture and biotechnology. However, it is difficult to LS Linsmaer and Skoog medium (1965)
develop appropriate breeding material by in vitro cultiva- C Dumas de Vaulx et al. induction medium
tion in this species. Haploid plant production is useful in (1981)
the breeding programs to facilitate recovery of recessive R Dumas de Vaulx et al. regeneration
mutations and unique genetic recombinations. In embryo- medium (1981)
genesis, haploid formation from pollen in anther culture is Cm Sibi et al. induction medium (1979)
a scientifically advanced, but controversial system. Various Rm Sibi et al. regeneration medium (1979)
techniques for haploid plant regeneration are used to CP Chambonnet induction medium (1988)
establish an efficient double haploid production method. R1, R2 Chambonnet regeneration media (1988)
The purpose of this article is to summarize, through com- 2.4-D 2.4-Dichlorophenoxyacetic acid
parison, results in pepper anther culture, problems associ- BA 6-Benzyladenine
ated with work in this field, and the influence of critical IAA Indole-3-acetic acid
factors for successful embryo formation and plantlet TDZ (DROPP) Thidiazuron
development. NAA a-Naphtaleneacetic acid
EDTA Ethylenediaminetetraacetic acid
Keywords Anther culture
Genotype
Pepper
Plant
growth regulators
Nutrient media
Thermal shock

Abbreviations
MS Murashige and Skoog basal medium Introduction
(1962)
B5 Gamborg et al. basal medium (1968) Pepper (Capsicum annuum L.) is an important vegetable
crop grown in temperate and tropical regions of the world.
This fact is due to the high biological value of the fruits
Communicated by A. K. Kononowicz. (high content of dry substance, vitamin C and B-complex,
minerals, essential oils, carotenoids, etc.) and their various
T. Irikova
kinds of utilization in the culinary and food industry of
Department of Developmental Biology,
Plovdiv University Paissii Hilendarski, different countries.
24 Tzar Assen Str., 4000 Plovdiv, Bulgaria Conventional breeding in pepper is a long-term and
labor-consuming process due to uncontrolled pollination
S. Grozeva
V. Rodeva (&)
and the necessity of isolation for prevention of genetic
Maritsa, Vegetable Crops Research Institute,
32 Brezovsko Shosse Str., 4003 Plovdiv, Bulgaria degeneration of breeding material. This may be overcome
e-mail: velirod@yahoo.com through in vitro methods for haploid plant production.

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1560
In pepper, haploid technology includes induction and
regeneration of haploid embryos from anthers or micro-
spore culture. Plant regenerants with a single chromosome
set, originating from microspores of in vitro cultivated
anthers, are ideal for genetic analysis due to the variety of
possible expressions of the genetic makeup (Atanassov
et al. 1995; Steinitz et al. 1999; Zagorska et al. 2002;
Arnedo Andres et al. 2004; Nervo et al. 2007; Pauk et al.
2010). The doubling of the haploid genome results in fully
homozygotic lines in shortened period which is important
for creation of genetic diversity and a base of new varieties
with higher quality. As reported in the most published
papers pepper embryogenesis is often limited due to diffi-
culties with low efficient system for plant regeneration and
developing of the shoots to normal plantlets (Nowaczyk
and Kisiala 2006; Kothari et al. 2010). According to dif-
ferent authors, the reasons for this inefficiency are from
various nature.
This review will describe the experience, the results and
knowledge achieved in pepper anther cultivation on the
base of internationally published data. The information is
useful for future research on initiation of embryogenesis
and regeneration of fertile double haploid plants aimed to
acceleration of the breeding programs.
Pepper anther culture
The first successful plant regeneration from C. annuum
anthers was reported by Wang et al. (1973) in China and
George and Narayanaswamy (1973) in India, and a year
later in C. frutescens by Novak (1974) in Poland. This was
followed by identification of a number of factors influ-
encing enhancement of pollen production and making the
process more efficient (Sibi et al. 1979; Dumas de Vaulx
et al. 1981; Dumas de Vaulx 1990).
Growing conditions and donor plant age
Anthers for culture can be obtained from field grown plants
or those grown under protected culture. The induction of
embryogenesis in pepper in vitro is influenced by condi-
tions affecting development of donor plants as well as their
physiological state (Bajaj 1990; Gonzalez-Garcia 2002).
Kristiansen and Andersen (1993) determined that donor
plant age, air temperature and photoperiod greatly influ-
ence the number of embryos developed in pepper anther
culture. Better plant embryo production occurs at an
average daily temperature of 26.4 0.4C, but poor
embryo induction occurs at 16 and 30C. Ltifi and Wenzel
(1994) reported differences in the ability to induce
embryogenesis between eight varieties, when donor plants
were grown at temperatures of 10 and 25C. According to
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Acta Physiol Plant (2011) 33:15591570
Buyukalaca et al. (2004), anthers from greenhouse grown
plants produce more embryos as compared to anthers from
plants grown under field conditions. There were also dif-
ferences in the anther culture response of pepper varieties
that were season dependent (Matsubara et al. 1998; Ercan
et al. 2006). The number of clear, sunny days and the
average monthly temperature during the vegetation period
of the donor plants influenced induction of embryogenesis
in anther culture (Rodeva and Cholakov 2006).
Growth conditions of the donor plants play a significant
role in determining the androgenic response of various
genotypes. In recent years, emphasis has been placed on
the refinement of the environment conditions. Haploid
induction ability and microspore responsiveness proved to
be almost twice at high in plants raised under optimal
artificial conditions in phytotron chambers when compared
with plants conventionally cultured in the greenhouse
(Mityko and Gemes 2006) and obviously is better to be
used in the development of more efficient laboratory pro-
tocol. Donor plant age is another factor influencing
embryogenesis. Powell (1990) and Reinert and Bajaj
(1992) reported that anthers excised from buds of the first
inflorescences possesses higher embryogenic potential.
Kristiansen and Andersen (1993) reported that there were
fewer responding anthers in older donor plants and lack of
embryogenesis in anthers from plants that were older than
12 weeks. However, anthers collected from older plants
have sufficient embryogenic potential when the optimal
developmental stage is used (Ercan et al. 2006). Rodeva
and Cholakov (2006) did not find a dependence between
embryogenesis induction, embryo yield and plant age. The
plant organism is a complex system with different and very
specific reactions to the environment in the course of the
growth which must be preliminary well studied by the
researchers because the optimal environment and plant age
seems to be variable and depend on the specificity of the
genotype. The analyzing of microspore viability in differ-
ent periods of donor plant growth could be helpful in
determining season influence and successful androgenic
induction.
Genotype of the donor plant
The genotype is the most important and often limiting factor
in the pepper androgenic reaction (Comlekcioglu et al. 2001;
Rodeva 2001; Wang and Zhang 2001; Rodeva et al. 2004;
Koleva-Gudeva et al. 2007; Liu et al. 2007). Some pepper
genotypes are recalcitrant towards induction of androgene-
sis and formation of haploid regenerants. For lines and F1
hybrids frequency of direct embryogenesis varies from 0.5
to 75 embryoids per 100 cultivated anthers, and is genotype
dependent (Qin and Rotino 1993; Ltifi and Wenzel 1994;
Mityko et al. 1995; Koleva-Gudeva et al. 2007). After anther
Acta Physiol Plant (2011) 33:15591570
cultivation of more than 500 varieties, breeding lines and F1
hybrids, Mityko and Fari (1997) found substantial differ-
ences in the androgenic response. Anther forming embryos
were reduced in the order wax type [ dark green type [
tomato shape [ hot pepper (from 75 to 0 plants per 100
anthers).
According to Kristiansen and Andersen (1993), opti-
mization of growth conditions of donor plants does not
eliminate genotype influence. Genotype has a significant
effect on induction of embryogenesis and on subsequent
development of embryos to plant regenerants but also
influences the correlation between obtained haploid and
spontaneously diploidized regenerants (Dolcet-Sanjuan
et al. 1997; Mityko and Fari 1997). Nowaczyk et al. (2009)
demonstrated clear differences in the effectiveness of
androgenesis between the pepper hybrid forms as well as
among individual plants of tested genotypes. There are
differences in embryogenic potential among genotypes
(Rodeva et al. 2004, 2006). The capacity for androgenic
induction in anther culture is higher if donor plants are
derived from successfully diploidized haploid lines, but
reactions of heterogenic initial breeding material are dif-
ficult to predict (Venczel and Gemesne Juhasz 1998).
Obviously, the data prove that the ability of plants to
produce androgenic embryos with microspore origin
strongly depends on the genetic specificity of the cultivars
and must be evaluated before starting the experiments.
Overcoming the problem with the recalcitrant nature of
some pepper genotypes is possible after hybridization with
much more responsive ones for transfer of genes deter-
mining the embryogenic reaction or by looking for differ-
ent physical chemistry conditions for anther cultivation.
Microspore developmental stage
According to Bajaj (1984) pollen at the uninucleate stage
before or during the first mitosis is sensitive to external stress
that could hinder microspore embryogenesis. In different plant
species, it is recommended that uninucleate microspores are
most appropriate for induction of embryo formation in anther
culture (Heberle-Bors 1989; Vicente et al. 1991; Telmer et al.
1992; Pretova et al. 1993; Mityko and Fari 1997). According to
Supena et al. (2006), the determining factor for successful
anther culture is the selection of flower buds containing over
50% microspores in the late-uninucleate phase. The early
binucleate microspore stage is also amenable to androgenesis
induction (Gonzalez-Melendi et al. 1995, 1996a; Kao and
Horn 1999). Kim et al. (2004, 2008) reported that anthers
containing over 75% microspores in early binucleate phase
are optimal for embryo production in isolated microspore cul-
tures. Lantos et al. (2009) found that the anthers containing
80% late uninucleate and 20% early binucleate microspores
improve embryogenesis in microspore culture.
1561
Substantial differences concerning microspore devel-
opmental stage most suitable for embryogenesis induction
occur between plant species (Gresshoff and Doy 1972;
Touraev and Heberle-Bors 2003; Bal and Abak 2007), and
between varieties of a single species (Ahmadian et al.
1998; Kao and Horn 1999). In addition to the develop-
mental stage, the ability of microspores to survive under in
vitro conditions is important. Microspore survival varies
from 2 to 75% depending on the genotype (Gemesne et al.
1998). Of course, many factors influence the process at this
momentthe wounding of the microspores, convenient
composition of the culture medium and light intensity, the
shock temperatures.
The size and morphology of flower buds can be used as
an indirect indication for determining microspore stage
development (Sibi et al. 1979). Microspores in the process
of first pollen mitosis occur in flower buds with corolla
petals and calyx sepals of equal length or with petals
slightly longer than sepals (O zkum and Tipirdamaz 2002;
Nowaczyk and Kisiala 2006). Anthers are distinguished by
the presence of a light anthocyan tinge in the distal end
(Comlekcioglu et al. 2001). These indirect characteristics
are not applicable in all varieties because of significant
differences in bud morphology between genotypes (Rodeva
2001; Rodeva et al. 2007; Irikova 2008).
For development of an efficient experimental anther
culture system, the late uninucleate and early binucleate
stages are most suitable. It is advisable that in vitro
androgenesis always should start with precise cytological
analysis so that flower bud size and morphology corre-
spond to the appropriate developmental stages for anther
cultivation.
Culture media
There are several widely used basal media, and media with
relatively limited application (Table 1). Researchers gen-
erally prefer the basal media of Dumas de Vaulx et al.
(1981) and Murashige and Skoog (1962) that exhibit effi-
ciency of haploid plant production. The final medium
composition is modified mainly by changing the concen-
tration and combination of plant growth regulators or other
additives.
Sucrose is a commonly used carbohydrate for in vitro
cultivation of pepper anthers mostly in a 3% concentration.
For improving efficiency of microspore embryogenesis
carbohydrate source, or its quantity, in the media is mod-
ified by increasing sucrose concentration (Morrison et al.
1986b; Binzel et al. 1996; Supena et al. 2006) or using
maltose as the carbohydrate (Dolcet-Sanjuan et al. 1997;
Gemesne et al. 2000; Gyulai et al. 2000; Supena et al.
2006). Dolcet-Sanjuan et al. (1997) explain the stimulatory
effect of maltose on embryogenesis due to delaying
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Table 1 Culture media for anther culture of pepper and outcome of use of the medium
Type of medium Finding References

Basal Supplements

CR 0.01 mg/l kinetin 540 plants per 100 cultured anthers Dumas de Vaulx et al. (1981)
0.01 mg/l 2.4-D Average 2.1 embryos per 100 cultured Kristiansen and Andersen (1993)
anthers Ltifi and Wenzel (1994)
15.56% embryos per 100 anthers Mityko and Fari (1997)
0178.2% pollen embryo induction Dolcet-Sanjuan et al. (1997)
11.9 embryos per 100 flowers Rodeva et al. (2004)
04.0% anthers with embryos Irikova and Rodeva (2005)
010.96% embryogenic anthers Supena et al. (2006)
00.2 plants per bud Koleva-Gudeva (2003a, b, 2007)
033.66% embryogenic anthers Rodeva et al. (2007)
017.39% embryogenic anthers
0.5 mg/l BA 268 embryos from 2,937 cultured buds Qin and Rotino (1993)
0.5 mg/l 2.4-D
0.01 mg/l kinetin 4.8% frequency of plant development; Gyulai et al. (2000)
0.01 mg/l 2.4-D 144 regenerants per 3,000 anthers Gemesne et al. (2000)
3% maltose On an average 100 responsive anthers
produced 18 2.5 pollen embryos
5.0 mg/l kinetin 1.17% anthers with embryos Ellialtioglu et al. (2001)
5.0 mg/l 2.4-D
1% activated charcoal
200 ml/l carrot extract
MS 1% activated charcoal Highest frequency of induced direct Pandeva and Zagorska (1986),
embryogenesis Pandeva et al. (1990)
0.1 mg/l kinetin 02.6% embryogenic anthers Matsubara et al. (1998)
0.004 mg/l 2.4-D 011.12% embryogenic anthers Irikova and Rodeva (2004)
011% anthers with embryos, 013.8% Rodeva et al. (2006)
embryo structures, 02.3% obtaining
regenerants
4.0 mg/l NAA 2.28% anthers with embryo Ellialtioglu et al. (2001)
1.0 mg/l BA
4.0 mg/l NAA No embryo obtained Comlekcioglu et al. (2001)
0.1 mg/l BA
0.25% activated charcoal
4.0 mg/l NAA 7.550% embryogenic flower buds
0.1 mg/l BA
0.25% activated charcoal
10 mg/l AgNO3
2.0 mg/l IAA 7 regenerated plantlets Gonzalez-Garcia (2002)
0.3 mg/l BA
4.0 mg/l NAA 12.5% mean androgenic embryo zkum and Tipirdamaz (2002)
O
1.0 mg/l BA production
0.25% activated charcoal
0.2 mg/l kinetin 00.83% embryogenic anthers; Irikova and Rodeva (2004)
2.0 mg/l IAA
2.0 mg/l NAA 09.68% embryogenic anthers
2.0 mg/l BA
Without plant growth regulators 011.54% embryogenic anthers
2% sucrose 0.5 plants per bud (modifications by Supena et al. (2006)
0.5% activated charcoal Johansson et al. (1982) and Custers
et al. (1999))
1.0 mg/l kinetin No embryos, only callus formation Koleva-Gudeva (2003a),
0.01 mg/l 2.4-D Koleva-Gudeva et al. (2007)
0.001 mg/l IAA
0.1 mg/l kinetin 0.327.6% embryogenic anthers Rodeva et al. (2007)
0.3 mg/l 2.4-D
Vitamin B5

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Acta Physiol Plant (2011) 33:15591570 1563

Table 1 continued
Type of medium Finding References

Basal Supplements

CmRm 2.0 mg/l kinetin 3 plants per 100 anthers Sibi et al. (1979)
2.0 mg/l 2.4-D 08.9% anthers with embryo structures; Rodeva (2001)
03.6% anthers with regenerants
07.14% embryogenic anthers Irikova and Rodeva (2005)
2.0 mg/l kinetin 08.34% embryogenic anthers Irikova and Rodeva (2005)
2.0 mg/l 2.4-D
5.0 mg/l AgNO3
10-8 M 2.4-D 439% anthers with embryos (over 100 Morrison et al. (1986b)
2% activated charcoal embryos, produced 13 plants in
double-layer Rm by Johansson et al.
6% sucrose 1982)
EDTA by MS
CPR1, R2 01.2% anthers with embryos Simeonova et al. (1990)
00.15% plantlets
0.5% activated charcoal 4.1% embryogenic anthers Nowaczyk et al. (2006)
5.0 mg/l AgNO3
B5a No embryos, only callus formation Mythili and Thomas (1999)
Na 5.0 mg/l kinetin 4.4% frequency of embryo formation Boyaci (2001)
5.0 mg/l 2.4-D but embryos could not survive
1% activated charcoal
1.0 mg/l kinetin No embryos, only callus formation Koleva-Gudeva and Spasenoski
0.01 mg/l IAA (2002), Koleva-Gudeva (2003a),
Koleva-Gudeva et al. (2007)
NNa 2% maltose Mean 111.9 embryos per 100 flowers Dolcet-Sanjuan and Claveira
0.5% activated charcoal (double layer medium) (1994), Dolcet-Sanjuan et al.
(1997)
0.55.0 lM kinetin No embryos, only callus formation Mythili and Thomas (1999)
0.520 lM 2.4-D
5.0 lM IAA
2.5 lM zeatin Mean 40.8 embryos, 1 plant per bud Supena et al. (2006)
5.0 lM IAA (double layer medium)
2% maltose
0.5% activated charcoal
0.01 mg/l kinetin No embryos, only callus formation Koleva-Gudeva and Spasenoski
0.001 mg/l 2.4-D (2002), Koleva-Gudeva et al.
(2007)
LSa 3.0 mg/l kinetin No embryos, only callus formation Koleva-Gudeva and Spasenoski
1.0 mg/l IAA (2002), Koleva-Gudeva (2003a),
Koleva-Gudeva et al. (2007)
a
Basal medium with limited application

conversion to glucose, and avoiding the inhibitory effect of
other sugars on the microspore development.
Plant growth regulators
Several of these compounds (Table 1) are used to stimulate
microspore embryogenesis. Kinetin and 2.4-dichlorophe-
noxyacetic acid (2.4-D) (1:1) are used in concentrations
from 0.01 to 5.0 mg/l, or kinetin is used at 0.1 mg/l and
2.4-D at 0.004 mg/l (Sibi et al. 1979; Dumas de Vaulx et al.
1981; Chambonnet 1988; Yoon et al. 1991; Matsubara et al.
1998; Prayantini 2006). Lowering the concentrations of the
both plant growth regulators from 0.01 to 0.05 mg/l stim-
ulates embryogenesis in pepper; when used at 0.08 mg/l
callusogenesis is stimulated (Gemesne et al. 1998). Qin and
Rotino (1993) found that 6-benzyladenine (BA) (0.6 mg/l)
and the combination of BA with 2.4-D promote embryo-
genesis in some recalcitrant genotypes. Indole-3-acetic acid
(IAA) is essential for induction of cell division in pollen
when haploid morphogenesis is desired, but numbers of
viable plantlets are not more than 0.1% per several hundred
cultivated anthers (George and Narayanaswamy 1973).
When used alone, or in combination with other plant growth
regulators, such as thidiazuron (TDZ, DROPP), kinetin or
2.4-D, IAA stimulates callusogenesis (Sukhanov et al.
1977; Pandeva et al. 1990; Mythili and Thomas 1995;
Koleva-Gudeva and Spasenoski 2002; Koleva-Gudeva
2003b). Callusogenesis is also observed when cytokinins

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1564
are combined with a-naphtaleneacetic acid (NAA) (Park
et al. 1992; Wang et al. 2004). Low concentrations of NAA
(up to 0.1 mg/l) or its combination with kinetin, 2.4-D and
BA, positively increase anther embryogenesis reaction
(Yoon et al. 1991; Ellialtioglu et al. 2001; O zkum et al.
2001; Ozkum and Tipirdamaz 2002; Kim et al. 2004).
Gonzalez-Garcia (2002) and Supena et al. (2006) demon-
strated that 2.0 mg/l IAA combined with BA (0.3 mg/l),
and 5 lM IAA with zeatin (2.5 lM) are suitable for initi-
ating microspore embryogenesis. The different androgenic
responsiveness is due to genetic predetermination of the
genotype and the ratio between endogenous and exogenous
plant growth regulators.
Media without auxins or containing different concen-
trations of kinetin for further development of embryos to
plant regenerants were studied (Mityko et al. 1995;
Venczel et al. 1996; Barcaccia et al. 1999; Boyaci 2001;
Koleva-Gudeva et al. 2007). The successful regeneration in
media without phytohormones was reported by Park et al.
(1992), Matsubara et al. (1998), Ellialtioglu et al. (2001),
Prayantini (2006), and Rodeva et al. (2006). The process of
embryo conversion to plantlets is influenced by genotype,
basal media composition, perhaps the physical conditions
of cultivation and interactions between them.
Supplements to the culture media
Activated charcoal absorbs substances, including phenols
and abscisic acid inhibitory to microspore embryogenesis
from the agar and the environment of culture vessel as well
as ingredients with negative effects produced by anther
tissues (Kohlenbach and Wernicke 1978; Johansson 1983;
O zkum and Tipirdamaz 2002). Activated charcoal can also
absorb FeEDTA, pyridoxine, biotin, thiamine, folic acid,
nicotine acid and hormone-supporting plant growth
released from anther tissues (Weatherhead et al. 1979;
Johansson 1983; Johansson et al. 1990; Vagera 1990).
Carbon stimulates embryogenesis in pepper and prolongs
vitality and productivity of anthers by inhibiting callus
proliferation, but inhibits growth of embryos and devel-
opment of regenerants (Vagera and Havranek 1985; Boyaci
2001). Charcoal in media at concentrations from 0.25 to
2% (Morrison et al. 1986b; Dolcet-Sanjuan and Claveira
1994; O zkum et al. 2001; O zkum and Tipirdamaz 2002;
Supena et al. 2006), or in combination with carrot extract
stimulates embryogenesis (Vagera 1984; Pandeva and
Zagorska 1986; Pandeva et al. 1990) (Table 1). Ellialtioglu
et al. (2001) observed increased embryo production on
medium C and decreased on medium MS (Murashige and
Skoog 1962) supplemented with carrot extract and acti-
vated charcoal.
Silver nitrate is known as an inhibitor of ethylene
released during in vitro plant tissue cultivation (Beyer 1976;
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Acta Physiol Plant (2011) 33:15591570
Santana-Buzzy et al. 2006). Beneficial effects of AgNO3
with activated charcoal on efficiency of androgenesis in
pepper anther culture have been reported (Nervo et al. 1995;
Comlekcioglu et al. 2001; Caglar et al. 2004; Nowaczyk and
Kisiala 2006; Nowaczyk et al. 2006). Luz et al. (1996, 1997)
observed improved embryogenic induction in medium
containing only AgNO3, but inhibition of the processes
when combined with activated charcoal. Irikova and Rodeva
(2005) reported that effects of silver nitrate depend on
the basal medium. Buyukalaca et al. (2004) obtained the
most embryos at a concentration of 15 mg/l AgNO3
(45.7 embryos per 100 anthers). Combining charcoal with
carrot extract and silver nitrate can promote embryo for-
mation, but the results are also dependent on basal medium
composition and cultivation period.
Other additives (antibiotics as Ampicillin, Rifocin,
Streptomycin, Neomycin and Acetylsalicylic and Ascorbic
acids) can be added to the medium to increase anther
culture efficiency, but their influence on embryo induction
is not well known and more experiments are needed to
prove it (Gomez and Chambonnet 1992; Luz et al. 1997;
Gemesne et al. 1998; Wang et al. 2004).
Temperature stress treatment
Embryo programming of the microspore requires stress
(Ahmadian et al. 1998; Barany et al. 2005; Jacquard et al.
2006). To switch from the microspore gametophyte to
sporophyte embryo stage, a physical, i.e., low or high
temperature, or osmotic stress, or a physiological, i.e.,
starvation stress can be applied (Maraschin et al. 2005;
Koleva-Gudeva et al. 2009).
The response to temperature on flower buds and anthers
vary (Table 2). When 4C temperature for 48 h was applied
as pre-treatment to flower buds embryogenesis was
improved and 13 plants per 100 anthers obtained (Sibi et al.
1979). Cold pretreatment of flower buds from 24 to 100 h
before excising anthers for culture stimulated the androge-
netic response (Morrison et al. 1986a, b; Supena et al. 2006).
However, it has also been reported that there are no signif-
icant effects of flower bud cold pretreatment (Vagera and
Havranek 1985; Munyon et al. 1989; O zkum et al. 2001;
Kim et al. 2005). Ozkum and Tipirdamaz (2002) found that
reduced embryo formation after cold pretreatment due to
callus induction on the explants. This means that in different
experimental conditions and genotypes, the reaction of the
anthers could be different and often opposite.
Dolcet-Sanjuan et al. (1997) stimulated embryogenesis
by cultivating anthers for a week at 7C in the dark. Supena
et al. (2006) increased embryogenic potential by treating
anthers with 9C during the first week of cultivation, but
Koleva-Gudeva et al. (2007) did not report a similar effect
(Table 2).
Acta Physiol Plant (2011) 33:15591570 1565

Table 2 Temperature effect on induction of pepper microspore embryogenesis in vitro

Temperature Applied to Finding References

Low (C)
4 Flower buds Stimulate embryogenesis Sibi et al. (1979), Morrison et al.
No positive effect (1986a, b), Supena et al. (2006)
Vagera and Havranek (1985), Munyon
zkum et al. (2001),
et al. (1989), O
zkum and Tipirdamaz (2002), Kim
O
et al. (2005)
7 Anthers Stimulate embryogenesis Dolcet-Sanjuan et al. (1997)
No positive effect Koleva-Gudeva et al. (2007)
9 Anthers Stimulate embryogenesis Supena et al. (2006)
High (C)
29 Anthers Stimulate embryogenesis Phillips et al. (1984),
Ellialtioglu et al. (2001)
35 Anthers Stimulate embryogenesis Sibi et al. (1980), Dumas de Vaulx et al.
(1981, 1982), Morrison et al. (1986b),
Koleva-Gudeva (2003a), Prayantini
(2006)

Sibi et al. (1980) reported that application of high
temperature (35C) during the first 2 days of anther culti-
vation more efficiently stimulates embryogenesis when
compared with cold (4C) pre-treatment of flower buds.
A highly effective method for embryo induction was
developed by Dumas de Vaulx et al. (1981, 1982). They
exposed isolated anthers to heat shock stress (35C) in the
dark for 8 days and had a better response when compared
with a shorter cultivation at the same temperature. The
efficiency of this shock treatment on anthers was later
confirmed by others (Koleva-Gudeva 2003a; Prayantini
2006). Morrison et al. (1986b) modified the method of
Dumas de Vaulx et al. (1981) by increasing the anther
cultivation period (to 12 days), observing that treatment
with 35C was better compared with incubation at 25C
(Table 2).
Incubation of anthers at 28.5 to 29C under continuous
diffused light increased frequency of microspore embryo
development (Phillips et al. 1984; Ellialtioglu et al. 2001).
Mak and Maheswary (1994) found that the optimal
androgenic response under various cultivation conditions is
genotype dependent. With cold bud pretreatment (Irikova
et al. unpublished) embryo yield depends on genotype and
some varieties produce more embryos after 72 h at 4C
than at 25C.
Several reports discuss how temperature affects cellular
mechanisms (Hepler and Palevitz 1974; Bajaj 1978;
Touraev et al. 1997) and the effect on genetic expressions
that switch microspores to embryogenesis (Gonzalez-
Melendi et al. 1995, 1996a, b; Testillano et al. 2000a, b;
Barany et al. 2001). The critical point for application of
temperature shock treatments is in late uninucleate and
early binucleate stages of pollen development, a point
where microspores deviate from normal male development
in the gametophyticsporophytic transition in the mother
pollen cells.
The stimulation of embryo induction with temperature
shock is extremely important, especially in genotypes with
poor response. It is obviously, from summarized results,
that the factors identified as critical for inducing the
embryogenesis in pepper anthers must be combined care-
fully with all other conditions mentioned above depending
on the specific genotype answer.
Cytological studies of microspore embryogenesis
In pepper anther culture, a large number of the pollen
grains in the anther sac remain in a uninuclear stage and
gradually degenerate, but some undergo division and
develop into a multicell mass which either to form callus or
differentiate to development of regenerants (Wang et al.
1973; Pandeva and Zagorska 1986). According to Ahma-
dian et al. (1998), the path for development of pollen is
genetically controlled. After stress, microspores symmet-
rically divide and bicellular embryos, similar in size and
cytoplasm organization, are formed. Subsequent cell divi-
sions lead to the formation of multicellular embryos with
smaller cells which are still surrounded by a pollen wall.
According to Barcaccia et al. (1999), two consecutive
mitoses take place in uninuclear microspores producing
four vegetative nuclei. Rarely tri-nuclear microspores
originate with two vegetative and one generative nucleus.
In 810 days of cultivation, there are pro-embryos with
over 1012 nuclei still enclosed in the exine. Because the
generative nucleus never undergoes a subsequent division,

123
1566
androgenesis in pepper probably follows the so-called B
pathway, which happens when the embryos are formed
only from two symmetric vegetative nuclei resulting from
the first pollen mitosis (Sunderland 1973). The cytological
process of early microspore embryogenesis was described
by Testillano et al. (2000a), Barany et al. (2001, 2005) and
Kim et al. (2004, 2008).
Ploidy level of regenerants
Different approaches are applied for determining regener-
ant ploidy level. Vagera and Havranek (1985) observe
about 10% of androgenic pepper plants were completely
fertile, with normal fruits and without morphological
changes in their progeny. The remaining androgenic
regenerants were sterile, with narrow leaves, underdevel-
oped fruits and phenotypically corresponded to haploid
Capsicum types. Dolcet-Sanjuan et al. (1997) differentiate
the haploid from the diploid plants by the presence of
narrow leaves, shorter internodes and reduced vigor.
Wang et al. (1973) determined the chromosome number
in callus and apical root cells of regenerants, and found the
haploid number (n = 12). In 24 androgenic regenerants,
Sibi et al. (1979) found 20 haploid, 2 diploid and 2 triploid
plantlets. The high frequency of diploids or haploiddip-
loid chimeras among the plant regenerants was reported
(Dumas de Vaulx et al. 1981). Mityko et al. (1995) reported
that the ratio of haploid and diploid regenerants is 1:1.
Barcaccia et al. (1999) found spontaneous diploidization in
47.83% of haploids. Ploidy of regenerants by microscopic
analysis was determined by Phillips et al. (1984), Matsubara
et al. (1998) and Gemesne et al. (2000).
Qin and Rotino (1995) assumed that the ploidy level is
identified by chromosome number in mitotic cells of apical
roots, or in meiotic cells of microspores, but for a large
number of plants, this type of analyses is time consuming.
They developed an indirect determination of ploidy by
counting numbers of chloroplasts in guard cells of stomata
which is a rapid method for distinction of haploid from
diploid plants. Abak et al. (1998) agreed that this is a
reliable indication for specifying the ploidy level.
Determining the ploidy level of the pepper regenerants
became possible by measuring the quantity of DNA in
nuclei of cells from young leaves with flow cytometric
analysis (Dolezel et al. 1989; Lanteri et al. 2000). Mityko
and Fari (1997) found that a correlation of haploid to
doubled haploid regenerants depends on genotype. The
spontaneous level of haploid to doubled haploid varies
from 1:1 to 1:2 in large fruit pepper, and from 3:1 to 2:1 in
pepper used for spices. Gemesne et al. (1998, 2001a) dif-
ferentiated 6075% haploid plantlets, 3540% spontaneous
doubled haploids, 0.7% tetraploids and 1.0% aneuploids.
123
Acta Physiol Plant (2011) 33:15591570
Similar correlations among regenerate haploid (64.5%),
spontaneous doubled haploid (32.6%), tetraploid (0.6%),
and aneuploid (2.3%) plants were established by Gyulai
et al. (2000). The study of the ploidy level of the regen-
erants must be drew attention because the in vitro culti-
vation stimulate also cell division in the somatic tissue of
the anther wall.
Enzyme markers
To establish the origin, and the homo- and heterozygosity
of callus and regenerants obtained from anther culture,
enzyme markers are used (Phillips et al. 1984; Munyon
et al. 1989). Dolcet-Sanjuan and Claveira (1994) and
Dolcet-Sanjuan et al. (1997) found the microspore origin of
regenerants by electrophoretic analysis of isocitratdehy-
drogenase. Morrison et al. (1986b) established homozy-
gous regenerants through electrophoretic separation of
shikimatedehydrogenase and phosphoglucomutase.
Diploidization of the genome
Colchicine treatment at earlier stage in the formation of
plantlets, later on shoot tips, on fully developed haploid
plants or secondary axillary buds for diploidization of the
haploid genome and obtaining fertile homozygous forms
(Bajaj 1984; Chambonnet 1988; Caglar and Abak 1997;
Dolcet-Sanjuan et al. 1997). Mityko and Fari (1997) found
that after conventional colchicine treatment of pepper
plants only about 50% are successfully diploidizated; when
applied in vitro on young plants the efficient is higher (75%
diploidization). Mityko et al. (1998) added colchicine,
100500 mg/l, to the induction medium and treated anthers
for 2496 h. They reported that colchicine does not
improve the in vitro response in recalcitrant genotypes;
however, the increased frequency of doubled haploid
regeneration is slightly influenced depending on concen-
tration and duration of the colchicine treatment. Plantlets
with different ploidy level are not developed and fertility of
induced doubled haploids is as in spontaneously produced
double haploids. According to Gemesne et al. (2001a, b)
and Gemes (2006), 6 days of cultivation of 1-month old
haploid regenerants on medium containing colchicine
(0.04%) increased the percentage of doubled haploids up to
95%.
Conclusion
Anther culture of pepper is an attractive system for pro-
duction of haploid plantlets. However, an efficient protocol
for genotype-independent haploid induction is still lacking
Acta Physiol Plant (2011) 33:15591570
and the use of double haploids is limited. There is vari-
ability of results within treatments, likely due to genetics
differences and heterogeneity of donor plants providing
anthers. The potential of pepper genotypes that respond
with embryogenesis varies from good to poor and non-
responsive genotypes. Not all embryos formed in anthers
are able to regenerate into fully developed plants and not
all acclimatized plants are fertile. Critical factors for
embryo production and plantlet regeneration are donor
plants growth conditions, selection of flower buds with
microspores at the appropriate stage, stress, medium
composition, genetic background of genotypes and physi-
cal culture conditions.
The in vitro studies are still empirical and preliminary
experiments are important for establishing: which geno-
types should be used; basal media (induction medium C
and regeneration medium RDumas de Vaulx et al.
1981; MSMurashige and Skoog 1962); plant growth
regulators (kinetin and 2,4-D); developmental stage of
microspores (late uninucleate microspores); stress (4C
pretreatment or/and 35C treatment) and how the genetic
structure of certain genotypes is responsible for, and
involved in, activation or repression of microspore
developmental process.
In terms of plant breeding, the key for increased
regeneration efficiency during androgenesis will largely
depend on the control of two main developmental
switchesthe induction of microspore cell division and
their ultimate commitment to the embryogenic pathway.
Although there has been a considerable increase in the
amount of information concerning cellular and molecular
aspects involved in microspore embryogenesis induction
and embryo formation, still very little is known about the
mechanism behind the change of pathways from game-
tophytic to embryonic. Future efforts should focus on
further investigation of pollen development and refine-
ment of conditions facilitating embryo induction.
Microspore culture offers an alternative means of
developing haploid and double haploid plants because iso-
lated microspores avoid formation of callus and embryos
from somatic tissue and all regenerated plants are presum-
ably haploids or double haploids. Although promising data,
this technique is still difficult, time and labor consuming,
which are factors limiting its exploitation.
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