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Acta Physiol Plant (2011) 33:15591570

DOI 10.1007/s11738-011-0736-6


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

MS Murashige and Skoog basal medium Introduction
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: through in vitro methods for haploid plant production.

1560 Acta Physiol Plant (2011) 33:15591570

In pepper, haploid technology includes induction and Buyukalaca et al. (2004), anthers from greenhouse grown
regeneration of haploid embryos from anthers or micro- plants produce more embryos as compared to anthers from
spore culture. Plant regenerants with a single chromosome plants grown under field conditions. There were also dif-
set, originating from microspores of in vitro cultivated ferences in the anther culture response of pepper varieties
anthers, are ideal for genetic analysis due to the variety of that were season dependent (Matsubara et al. 1998; Ercan
possible expressions of the genetic makeup (Atanassov et al. 2006). The number of clear, sunny days and the
et al. 1995; Steinitz et al. 1999; Zagorska et al. 2002; average monthly temperature during the vegetation period
Arnedo Andres et al. 2004; Nervo et al. 2007; Pauk et al. of the donor plants influenced induction of embryogenesis
2010). The doubling of the haploid genome results in fully in anther culture (Rodeva and Cholakov 2006).
homozygotic lines in shortened period which is important Growth conditions of the donor plants play a significant
for creation of genetic diversity and a base of new varieties role in determining the androgenic response of various
with higher quality. As reported in the most published genotypes. In recent years, emphasis has been placed on
papers pepper embryogenesis is often limited due to diffi- the refinement of the environment conditions. Haploid
culties with low efficient system for plant regeneration and induction ability and microspore responsiveness proved to
developing of the shoots to normal plantlets (Nowaczyk be almost twice at high in plants raised under optimal
and Kisiala 2006; Kothari et al. 2010). According to dif- artificial conditions in phytotron chambers when compared
ferent authors, the reasons for this inefficiency are from with plants conventionally cultured in the greenhouse
various nature. (Mityko and Gemes 2006) and obviously is better to be
This review will describe the experience, the results and used in the development of more efficient laboratory pro-
knowledge achieved in pepper anther cultivation on the tocol. Donor plant age is another factor influencing
base of internationally published data. The information is embryogenesis. Powell (1990) and Reinert and Bajaj
useful for future research on initiation of embryogenesis (1992) reported that anthers excised from buds of the first
and regeneration of fertile double haploid plants aimed to inflorescences possesses higher embryogenic potential.
acceleration of the breeding programs. 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
Pepper anther culture 12 weeks. However, anthers collected from older plants
have sufficient embryogenic potential when the optimal
The first successful plant regeneration from C. annuum developmental stage is used (Ercan et al. 2006). Rodeva
anthers was reported by Wang et al. (1973) in China and and Cholakov (2006) did not find a dependence between
George and Narayanaswamy (1973) in India, and a year embryogenesis induction, embryo yield and plant age. The
later in C. frutescens by Novak (1974) in Poland. This was plant organism is a complex system with different and very
followed by identification of a number of factors influ- specific reactions to the environment in the course of the
encing enhancement of pollen production and making the growth which must be preliminary well studied by the
process more efficient (Sibi et al. 1979; Dumas de Vaulx researchers because the optimal environment and plant age
et al. 1981; Dumas de Vaulx 1990). seems to be variable and depend on the specificity of the
genotype. The analyzing of microspore viability in differ-
Growing conditions and donor plant age ent periods of donor plant growth could be helpful in
determining season influence and successful androgenic
Anthers for culture can be obtained from field grown plants induction.
or those grown under protected culture. The induction of
embryogenesis in pepper in vitro is influenced by condi- Genotype of the donor plant
tions affecting development of donor plants as well as their
physiological state (Bajaj 1990; Gonzalez-Garcia 2002). The genotype is the most important and often limiting factor
Kristiansen and Andersen (1993) determined that donor in the pepper androgenic reaction (Comlekcioglu et al. 2001;
plant age, air temperature and photoperiod greatly influ- Rodeva 2001; Wang and Zhang 2001; Rodeva et al. 2004;
ence the number of embryos developed in pepper anther Koleva-Gudeva et al. 2007; Liu et al. 2007). Some pepper
culture. Better plant embryo production occurs at an genotypes are recalcitrant towards induction of androgene-
average daily temperature of 26.4 0.4C, but poor sis and formation of haploid regenerants. For lines and F1
embryo induction occurs at 16 and 30C. Ltifi and Wenzel hybrids frequency of direct embryogenesis varies from 0.5
(1994) reported differences in the ability to induce to 75 embryoids per 100 cultivated anthers, and is genotype
embryogenesis between eight varieties, when donor plants dependent (Qin and Rotino 1993; Ltifi and Wenzel 1994;
were grown at temperatures of 10 and 25C. According to Mityko et al. 1995; Koleva-Gudeva et al. 2007). After anther

Acta Physiol Plant (2011) 33:15591570 1561

cultivation of more than 500 varieties, breeding lines and F1 Substantial differences concerning microspore devel-
hybrids, Mityko and Fari (1997) found substantial differ- opmental stage most suitable for embryogenesis induction
ences in the androgenic response. Anther forming embryos occur between plant species (Gresshoff and Doy 1972;
were reduced in the order wax type [ dark green type [ Touraev and Heberle-Bors 2003; Bal and Abak 2007), and
tomato shape [ hot pepper (from 75 to 0 plants per 100 between varieties of a single species (Ahmadian et al.
anthers). 1998; Kao and Horn 1999). In addition to the develop-
According to Kristiansen and Andersen (1993), opti- mental stage, the ability of microspores to survive under in
mization of growth conditions of donor plants does not vitro conditions is important. Microspore survival varies
eliminate genotype influence. Genotype has a significant from 2 to 75% depending on the genotype (Gemesne et al.
effect on induction of embryogenesis and on subsequent 1998). Of course, many factors influence the process at this
development of embryos to plant regenerants but also momentthe wounding of the microspores, convenient
influences the correlation between obtained haploid and composition of the culture medium and light intensity, the
spontaneously diploidized regenerants (Dolcet-Sanjuan shock temperatures.
et al. 1997; Mityko and Fari 1997). Nowaczyk et al. (2009) The size and morphology of flower buds can be used as
demonstrated clear differences in the effectiveness of an indirect indication for determining microspore stage
androgenesis between the pepper hybrid forms as well as development (Sibi et al. 1979). Microspores in the process
among individual plants of tested genotypes. There are of first pollen mitosis occur in flower buds with corolla
differences in embryogenic potential among genotypes petals and calyx sepals of equal length or with petals
(Rodeva et al. 2004, 2006). The capacity for androgenic slightly longer than sepals (O zkum and Tipirdamaz 2002;
induction in anther culture is higher if donor plants are Nowaczyk and Kisiala 2006). Anthers are distinguished by
derived from successfully diploidized haploid lines, but the presence of a light anthocyan tinge in the distal end
reactions of heterogenic initial breeding material are dif- (Comlekcioglu et al. 2001). These indirect characteristics
ficult to predict (Venczel and Gemesne Juhasz 1998). are not applicable in all varieties because of significant
Obviously, the data prove that the ability of plants to differences in bud morphology between genotypes (Rodeva
produce androgenic embryos with microspore origin 2001; Rodeva et al. 2007; Irikova 2008).
strongly depends on the genetic specificity of the cultivars For development of an efficient experimental anther
and must be evaluated before starting the experiments. culture system, the late uninucleate and early binucleate
Overcoming the problem with the recalcitrant nature of stages are most suitable. It is advisable that in vitro
some pepper genotypes is possible after hybridization with androgenesis always should start with precise cytological
much more responsive ones for transfer of genes deter- analysis so that flower bud size and morphology corre-
mining the embryogenic reaction or by looking for differ- spond to the appropriate developmental stages for anther
ent physical chemistry conditions for anther cultivation. cultivation.

Microspore developmental stage Culture media

According to Bajaj (1984) pollen at the uninucleate stage There are several widely used basal media, and media with
before or during the first mitosis is sensitive to external stress relatively limited application (Table 1). Researchers gen-
that could hinder microspore embryogenesis. In different plant erally prefer the basal media of Dumas de Vaulx et al.
species, it is recommended that uninucleate microspores are (1981) and Murashige and Skoog (1962) that exhibit effi-
most appropriate for induction of embryo formation in anther ciency of haploid plant production. The final medium
culture (Heberle-Bors 1989; Vicente et al. 1991; Telmer et al. composition is modified mainly by changing the concen-
1992; Pretova et al. 1993; Mityko and Fari 1997). According to tration and combination of plant growth regulators or other
Supena et al. (2006), the determining factor for successful additives.
anther culture is the selection of flower buds containing over Sucrose is a commonly used carbohydrate for in vitro
50% microspores in the late-uninucleate phase. The early cultivation of pepper anthers mostly in a 3% concentration.
binucleate microspore stage is also amenable to androgenesis For improving efficiency of microspore embryogenesis
induction (Gonzalez-Melendi et al. 1995, 1996a; Kao and carbohydrate source, or its quantity, in the media is mod-
Horn 1999). Kim et al. (2004, 2008) reported that anthers ified by increasing sucrose concentration (Morrison et al.
containing over 75% microspores in early binucleate phase 1986b; Binzel et al. 1996; Supena et al. 2006) or using
are optimal for embryo production in isolated microspore cul- maltose as the carbohydrate (Dolcet-Sanjuan et al. 1997;
tures. Lantos et al. (2009) found that the anthers containing Gemesne et al. 2000; Gyulai et al. 2000; Supena et al.
80% late uninucleate and 20% early binucleate microspores 2006). Dolcet-Sanjuan et al. (1997) explain the stimulatory
improve embryogenesis in microspore culture. effect of maltose on embryogenesis due to delaying

1562 Acta Physiol Plant (2011) 33:15591570

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
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)
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

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)
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.
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.
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)
Basal medium with limited application

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

1564 Acta Physiol Plant (2011) 33:15591570

are combined with a-naphtaleneacetic acid (NAA) (Park Santana-Buzzy et al. 2006). Beneficial effects of AgNO3
et al. 1992; Wang et al. 2004). Low concentrations of NAA with activated charcoal on efficiency of androgenesis in
(up to 0.1 mg/l) or its combination with kinetin, 2.4-D and pepper anther culture have been reported (Nervo et al. 1995;
BA, positively increase anther embryogenesis reaction Comlekcioglu et al. 2001; Caglar et al. 2004; Nowaczyk and
(Yoon et al. 1991; Ellialtioglu et al. 2001; O zkum et al. Kisiala 2006; Nowaczyk et al. 2006). Luz et al. (1996, 1997)

2001; Ozkum and Tipirdamaz 2002; Kim et al. 2004). observed improved embryogenic induction in medium
Gonzalez-Garcia (2002) and Supena et al. (2006) demon- containing only AgNO3, but inhibition of the processes
strated that 2.0 mg/l IAA combined with BA (0.3 mg/l), when combined with activated charcoal. Irikova and Rodeva
and 5 lM IAA with zeatin (2.5 lM) are suitable for initi- (2005) reported that effects of silver nitrate depend on
ating microspore embryogenesis. The different androgenic the basal medium. Buyukalaca et al. (2004) obtained the
responsiveness is due to genetic predetermination of the most embryos at a concentration of 15 mg/l AgNO3
genotype and the ratio between endogenous and exogenous (45.7 embryos per 100 anthers). Combining charcoal with
plant growth regulators. carrot extract and silver nitrate can promote embryo for-
Media without auxins or containing different concen- mation, but the results are also dependent on basal medium
trations of kinetin for further development of embryos to composition and cultivation period.
plant regenerants were studied (Mityko et al. 1995; Other additives (antibiotics as Ampicillin, Rifocin,
Venczel et al. 1996; Barcaccia et al. 1999; Boyaci 2001; Streptomycin, Neomycin and Acetylsalicylic and Ascorbic
Koleva-Gudeva et al. 2007). The successful regeneration in acids) can be added to the medium to increase anther
media without phytohormones was reported by Park et al. culture efficiency, but their influence on embryo induction
(1992), Matsubara et al. (1998), Ellialtioglu et al. (2001), is not well known and more experiments are needed to
Prayantini (2006), and Rodeva et al. (2006). The process of prove it (Gomez and Chambonnet 1992; Luz et al. 1997;
embryo conversion to plantlets is influenced by genotype, Gemesne et al. 1998; Wang et al. 2004).
basal media composition, perhaps the physical conditions
of cultivation and interactions between them. Temperature stress treatment

Supplements to the culture media Embryo programming of the microspore requires stress
(Ahmadian et al. 1998; Barany et al. 2005; Jacquard et al.
Activated charcoal absorbs substances, including phenols 2006). To switch from the microspore gametophyte to
and abscisic acid inhibitory to microspore embryogenesis sporophyte embryo stage, a physical, i.e., low or high
from the agar and the environment of culture vessel as well temperature, or osmotic stress, or a physiological, i.e.,
as ingredients with negative effects produced by anther starvation stress can be applied (Maraschin et al. 2005;
tissues (Kohlenbach and Wernicke 1978; Johansson 1983; Koleva-Gudeva et al. 2009).
O zkum and Tipirdamaz 2002). Activated charcoal can also The response to temperature on flower buds and anthers
absorb FeEDTA, pyridoxine, biotin, thiamine, folic acid, vary (Table 2). When 4C temperature for 48 h was applied
nicotine acid and hormone-supporting plant growth as pre-treatment to flower buds embryogenesis was
released from anther tissues (Weatherhead et al. 1979; improved and 13 plants per 100 anthers obtained (Sibi et al.
Johansson 1983; Johansson et al. 1990; Vagera 1990). 1979). Cold pretreatment of flower buds from 24 to 100 h
Carbon stimulates embryogenesis in pepper and prolongs before excising anthers for culture stimulated the androge-
vitality and productivity of anthers by inhibiting callus netic response (Morrison et al. 1986a, b; Supena et al. 2006).
proliferation, but inhibits growth of embryos and devel- However, it has also been reported that there are no signif-
opment of regenerants (Vagera and Havranek 1985; Boyaci icant effects of flower bud cold pretreatment (Vagera and
2001). Charcoal in media at concentrations from 0.25 to Havranek 1985; Munyon et al. 1989; O zkum et al. 2001;
2% (Morrison et al. 1986b; Dolcet-Sanjuan and Claveira
Kim et al. 2005). Ozkum and Tipirdamaz (2002) found that
1994; O zkum et al. 2001; O zkum and Tipirdamaz 2002; reduced embryo formation after cold pretreatment due to
Supena et al. 2006), or in combination with carrot extract callus induction on the explants. This means that in different
stimulates embryogenesis (Vagera 1984; Pandeva and experimental conditions and genotypes, the reaction of the
Zagorska 1986; Pandeva et al. 1990) (Table 1). Ellialtioglu anthers could be different and often opposite.
et al. (2001) observed increased embryo production on Dolcet-Sanjuan et al. (1997) stimulated embryogenesis
medium C and decreased on medium MS (Murashige and by cultivating anthers for a week at 7C in the dark. Supena
Skoog 1962) supplemented with carrot extract and acti- et al. (2006) increased embryogenic potential by treating
vated charcoal. anthers with 9C during the first week of cultivation, but
Silver nitrate is known as an inhibitor of ethylene Koleva-Gudeva et al. (2007) did not report a similar effect
released during in vitro plant tissue cultivation (Beyer 1976; (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
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

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

1566 Acta Physiol Plant (2011) 33:15591570

androgenesis in pepper probably follows the so-called B Similar correlations among regenerate haploid (64.5%),
pathway, which happens when the embryos are formed spontaneous doubled haploid (32.6%), tetraploid (0.6%),
only from two symmetric vegetative nuclei resulting from and aneuploid (2.3%) plants were established by Gyulai
the first pollen mitosis (Sunderland 1973). The cytological et al. (2000). The study of the ploidy level of the regen-
process of early microspore embryogenesis was described erants must be drew attention because the in vitro culti-
by Testillano et al. (2000a), Barany et al. (2001, 2005) and vation stimulate also cell division in the somatic tissue of
Kim et al. (2004, 2008). the anther wall.

Enzyme markers
Ploidy level of regenerants
To establish the origin, and the homo- and heterozygosity
Different approaches are applied for determining regener- of callus and regenerants obtained from anther culture,
ant ploidy level. Vagera and Havranek (1985) observe enzyme markers are used (Phillips et al. 1984; Munyon
about 10% of androgenic pepper plants were completely et al. 1989). Dolcet-Sanjuan and Claveira (1994) and
fertile, with normal fruits and without morphological Dolcet-Sanjuan et al. (1997) found the microspore origin of
changes in their progeny. The remaining androgenic regenerants by electrophoretic analysis of isocitratdehy-
regenerants were sterile, with narrow leaves, underdevel- drogenase. Morrison et al. (1986b) established homozy-
oped fruits and phenotypically corresponded to haploid gous regenerants through electrophoretic separation of
Capsicum types. Dolcet-Sanjuan et al. (1997) differentiate shikimatedehydrogenase and phosphoglucomutase.
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 Diploidization of the genome
in callus and apical root cells of regenerants, and found the
haploid number (n = 12). In 24 androgenic regenerants, Colchicine treatment at earlier stage in the formation of
Sibi et al. (1979) found 20 haploid, 2 diploid and 2 triploid plantlets, later on shoot tips, on fully developed haploid
plantlets. The high frequency of diploids or haploiddip- plants or secondary axillary buds for diploidization of the
loid chimeras among the plant regenerants was reported haploid genome and obtaining fertile homozygous forms
(Dumas de Vaulx et al. 1981). Mityko et al. (1995) reported (Bajaj 1984; Chambonnet 1988; Caglar and Abak 1997;
that the ratio of haploid and diploid regenerants is 1:1. Dolcet-Sanjuan et al. 1997). Mityko and Fari (1997) found
Barcaccia et al. (1999) found spontaneous diploidization in that after conventional colchicine treatment of pepper
47.83% of haploids. Ploidy of regenerants by microscopic plants only about 50% are successfully diploidizated; when
analysis was determined by Phillips et al. (1984), Matsubara applied in vitro on young plants the efficient is higher (75%
et al. (1998) and Gemesne et al. (2000). diploidization). Mityko et al. (1998) added colchicine,
Qin and Rotino (1995) assumed that the ploidy level is 100500 mg/l, to the induction medium and treated anthers
identified by chromosome number in mitotic cells of apical for 2496 h. They reported that colchicine does not
roots, or in meiotic cells of microspores, but for a large improve the in vitro response in recalcitrant genotypes;
number of plants, this type of analyses is time consuming. however, the increased frequency of doubled haploid
They developed an indirect determination of ploidy by regeneration is slightly influenced depending on concen-
counting numbers of chloroplasts in guard cells of stomata tration and duration of the colchicine treatment. Plantlets
which is a rapid method for distinction of haploid from with different ploidy level are not developed and fertility of
diploid plants. Abak et al. (1998) agreed that this is a induced doubled haploids is as in spontaneously produced
reliable indication for specifying the ploidy level. double haploids. According to Gemesne et al. (2001a, b)
Determining the ploidy level of the pepper regenerants and Gemes (2006), 6 days of cultivation of 1-month old
became possible by measuring the quantity of DNA in haploid regenerants on medium containing colchicine
nuclei of cells from young leaves with flow cytometric (0.04%) increased the percentage of doubled haploids up to
analysis (Dolezel et al. 1989; Lanteri et al. 2000). Mityko 95%.
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 Conclusion
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- Anther culture of pepper is an attractive system for pro-
ferentiated 6075% haploid plantlets, 3540% spontaneous duction of haploid plantlets. However, an efficient protocol
doubled haploids, 0.7% tetraploids and 1.0% aneuploids. for genotype-independent haploid induction is still lacking

Acta Physiol Plant (2011) 33:15591570 1567

and the use of double haploids is limited. There is vari- Ahmadian P, Testillano PS, Gonzalez P, Fadon B, Prestamo G,
ability of results within treatments, likely due to genetics Jimenez-Duran G, Risueno MC (1998) Cell biology of the pollen
developmental program and the induction of microspore
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with embryogenesis varies from good to poor and non- Avignon, France, pp 183186
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Abris JL, Palazon C, Luis Arteaga M, Gil Ortega R (2004)
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