Plant Cell, Tissue and Organ Culture 78: 201–208, 2004.

© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

201

Effect of sugars and amino acids on androgenesis of Cucumis sativus

H.G. Ashok Kumar & H.N. Murthy∗

Plant Tissue Culture Laboratory, Department of Botany, Karnatak University, Dharwad – 580003, Karnataka,
India (∗ requests for offprints: Fax: +91-836-2747884; E-mail: nmurthy60@yahoo.co.in)

Received 8 August 2003; accepted in revised form 8 December 2003

Key words: anther culture, cucumber, embryogenesis, plantlets

Abstract
The effects of sugars (sucrose, maltose, glucose and fructose) and amino acids (glutamine, glycine, arginine, as-
paragine and cysteine) on embryogenesis and plantlet regeneration from cultured anthers of Cucumis sativus L. cv.
Calypso and Green Long were studied. Type and concentration of sugar and amino acid influenced embryogenesis.
Among the different sugars tested, sucrose was the best for embryo induction with an optimal concentration of
0.25 M. Maximum of 72 and 80 embryos per 60 anthers of Calypso and Green Long, respectively, were induced
on embryo induction medium [B5 (Gamborg, Miller and Ojima (1968) Exp. Cell Res. 50: 151–158) supplemented
with 2.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 µM 6-benzyladenine (BA)] containing 0.25 M sucrose.
The addition of amino acids to the embryo induction medium improved embryo yield with a combination of
amino acids (glutamine, glycine, arginine, asparagine and cysteine of 1.0 mM each) giving the best response.
Embryo differentiation was achieved on B5 medium supplemented with 0.25 µM of α-naphthaleneacetic acid
(NAA), 0.25 µM kinetin (KN) and 0.09 M sucrose. Embryos were converted on B5 medium supplemented with
abscisic acid (ABA) (10 µM) and 0.09 M sucrose. Embryos that developed on B5 medium supplemented with a
combination of amino acids (glutamine, glycine, arginine, asparagine and cysteine of 1.0 mM each) exhibited the
highest plantlet regeneration frequency.

Abbreviations: ABA – abscisic acid; BA – 6-benzyladenine; B5 – Gamborg’s medium; 2,4-D – 2,4-

dichlorophenoxyacetic acid; KN – kinetin; NAA – α-naphthaleneacetic acid

Introduction et al., 2001). The production of haploids in cucum-

Cucumber (Cucumis sativus L.) is an important vege-
table crop and has been grown worldwide for food for
at least 3000 years. The world production of cucumber
has been estimated at 28.69 million tons annually, and
covered an area of 1.768 million hectares. In India,
cucumber is grown on 0.018 million hectares with a
total production of 0.117 million tons (Anonymous,
1999). Of two types of cucumber, pickling cucum-
ber (gherkin) production in India is estimated at 0.025
million tons annually and exported to U.S.A., Europe
and east Asian countries. The yield of the cucumber
is greatly affected by fungal, viral diseases and insect
pests (Malepszy, 1988). Anther culture is widely used
for practical breeding as a source of homozygous lines
(Bajaj, 1990), resistant lines to fungal diseases (Rizza
et al., 2002) and resistant lines to viral diseases (Niimi
ber through in vitro gynogenesis has been reported by
Sauton (1989), Niemirowicz-Szczytt and Dumas de
Vaulx (1989), Przyborowski and Niemirowicz-Szczytt
(1994) and Gémes Juhász et al. (1996, 2002). How-
ever, in all the cases the frequency of embryogenesis
and plant regeneration was low. Anther culture is
an efficient method for haploid induction compared
to ovary/ovule culture (Bajaj, 1990; Ferrie et al.,
1995). Lazarte and Sasser (1982) reported callus-
mediated embryogenesis/organogenesis from anther
cultures of cucumber. Ashok Kumar et al. (2003)
reported androgenesis in cucumber and studied the
effect of auxins, cytokinins and temperature pretreat-
ment of flower buds on embryogenesis from cultured
anthers.
Many factors influence androgenesis, including
plant genotype, the growing conditions of the donor
202
plants, the stage of the microspore, pre-treatment of
flower buds, the media and the culture conditions
(Bajaj, 1990). Medium composition is a key factor
influencing embryo/callus induction and subsequent
plant regeneration. Sugar is the source of carbon and
energy and also acts as an osmotic regulator in the
induction medium (Ferrie et al., 1995). The type and
concentration of sugars in the induction medium has
been found to influence androgenesis (Ferrie et al.,
1995). Sucrose, maltose, glucose and fructose are
the main carbohydrates used in culture media for an-
drogenesis with sucrose predominating (Ferrie et al.,
1995). For Triticum aestivum (Indrianto et al., 1999),
Secale cereale (Immonen and Anttila, 1998) maltose
was used as carbon source, while for strawberry
(Owen and Miller, 1996) glucose and for T. aestivum
(Chu et al., 1990) fructose proved to be the best carbon
sources. Further, sugar concentration has also been
shown to influence embryogenesis in many species
(Ferrie et al., 1995): 0.09 M sucrose for Quercus suber
(Bueno et al., 1997), 0.18 M sucrose for Oryza sativa
(Afza et al., 2000), 0.3 M sucrose or 0.28 M maltose
for Avena sativa and A. sterilis (Kiviharju and Pehu,
1998), 0.44 M sucrose for Cucurbita pepo (Metwally
et al., 1998).
Amino acids are known to improve embryogen-
esis and plant regeneration from cultured anthers
(Ferrie et al., 1995). The addition of an amino acid
mixture enhanced androgenesis in Hordeum vulgare
(Ouédraogo et al., 1998). Similarly, an amino acid
mixture improved androgenic green plant regeneration
in T. aestivum (Trottier et al., 1993). Amino acids
such as glutamine for T. aestivum (Indrianto et al.,
1999), thiamine and glycine for O. sativa (Guzmán
and Zapata, 2000) were supplemented in anther cul-
ture medium. Somatic embryo production was pro-
moted with the addition of amino acids in geranium
(Murthy et al., 1996).
The objective of the present investigation was to
determine the effect of sugars and amino acids on em-
bryogenesis and plantlet regeneration from cultured
anthers of cucumber.
Materials and methods
Plant material and surface sterilization of flower buds
The two cultivars of C. sativus L. used in these ex-
periments were Calypso and Green Long. The seeds
of Calypso and Green Long were procured from Ken
Agritech, Hubli, Karnataka, India and Mahyco veg-
etable seeds limited, Jalna, Maharastra, India, re-
spectively. Plants were grown in the experimental
plot, Department of Botany, Karnatak University,
Dharwad, Karnataka, India using standard agronomic
practices. Plants, 35–45 days old and grown during
the months of May–August were selected as donor
material. Flower buds were harvested when the mi-
crospores were at the mid to late uni-nucleate stage
(Figure 1A) and stored at 4 ◦ C for 2 days in dark
(Ashok Kumar et al., 2003). Pretreated flower buds
were washed in 1% (v/v) Laboline (detergent) and
0.5% Carbendazim (fungicide) for 10 min and surface
sterilized with 5% sodium hypochlorite solution for
20 min on an orbital shaker at 100 rpm. The final step
of sterilization was carried out in a horizontal lam-
inar air flow chamber by rinsing the flower buds twice
in sterile distilled water, followed by 0.1% mercuric
chloride solution for 5 min. Finally, the flower buds
were rinsed several times in sterile distilled water. An-
thers were removed from flower buds and inoculated
onto embryo induction medium.
Embryo induction, differentiation, maturation and
germination medium
B5 medium (Gamborg et al., 1968) supplemented
with 2.0 µM 2,4-D and 1.0 µM BA was used as
basal medium (Ashok Kumar et al., 2003). In ex-
periment 1, sucrose, maltose, glucose and fructose
were added individually at 0.05, 0.1, 0.15, 0.2, 0.25,
0.3, 0.35, 0.4, 0.45 or 0.5 M to B5 medium con-
taining 2.0 µM 2,4-D and 1.0 µM BA (embryo in-
duction medium-1). In experiment 2, amino acids
such as glutamine, glycine, arginine, asparagine
and cysteine at 0.5, 1.0, 2.0 or 5.0 mM were ad-
ded individually and also in combination at 1.0 or
2.0 mM each to B5 medium containing 2.0 µM 2,4-
D , 1.0 µM BA and 0.25 M sucrose (embryo induc-
tion medium-2). Responding cultures (globular stage
embryos/embryogenic calli with embryos) were sub-
cultured to B5 medium supplemented with 0.25 µM
NAA, 0.25 µM KN and 0.09 M sucrose (embryo dif-
ferentiation medium; Ashok Kumar et al., 2003).
Cotyledonary stage embryos were cultured onto em-
bryo maturation medium – B5 medium containing
0.09 M sucrose and supplemented with 10.0 µM ABA.
Mature embryos were isolated aseptically and cul-
tured onto B5 medium containing 0.09 M sucrose for
conversion (Ashok Kumar et al., 2003). The pH of
all media was adjusted to 5.8 using 0.1 N NaOH
 203

Figure 1. Embryogenesis and plantlet regeneration in cultured anthers of C. sativus L. (cvs. Calypso and Green Long). (A) Section of
anther of Calypso at the time of culture showing uninucleate microspores (bar = 0.01 mm). (B) A swollen anther of Calypso on induction
medium (B5 + 2.0 µM 2,4-D + 1.0 µM BA + 0.25 M sucrose) (bar = 1.2 mm). (C) Section of embryogenic anther of Calypso showing enlarged
microspore with pro-embryo-like structure (em: embryogenic microspore; nem: nonembryogenic microspore) (bar = 0.01 mm). (D) Globular
stage embryo of Calypso (bar = 0.8 mm). (E) Torpedo stage embryo of Calypso (bar = 1 mm). (F) Callus induction from anther of Green Long
(bar = 1.2 mm). (G) Cotyledonary stage embryo of Green Long (bar = 2.2 mm). (H) An anther derived plantlet of Calypso (bar = 1 cm).

or HCl and media were solidified with 0.8% agar
(Himedia, Mumbai, India). Medium was dispensed
into Petri dishes (20 ml per dish), Borosil rimless cul-
ture tubes (15 cm × 2.5 cm; 18 ml per culture tube) or
100 ml Borosil conical flasks (20 ml per flask). Cul-
ture tubes and flasks were plugged with nonabsorbent
cotton wrapped in cheesecloth or autoclavable caps.
The Petri dishes, culture tubes and culture flasks were
sterilized by autoclaving at 120 ◦ C for 20 min. Ther-
molabile compounds such as vitamins, amino acids
and ABA were filtered using sterilized membrane fil-
ters (Millipore; 0.45 µm) and added to the autoclaved
medium before aliquoting the medium into the culture
tubes/Petri dishes or conical flasks.
204
Culture conditions and acclimatization of plantlets
The cultures were incubated in the dark at 24 ± 2 ◦ C
for 2 weeks after inoculation and then at 24 ± 2 ◦ C and
16-h photoperiod of 40 µmol m−2 s−1 light provided
by cool white fluorescent tubes (Phillips, India). Well-
developed anther derived plantlets were removed from
cultures and washed in sterilized distilled water to
remove the traces of media. These plantlets were trans-
planted to plastic cups (7.5 cm × 8 cm) containing a
mixture of autoclaved coco-peat, sand and garden soil
(1:1:1). The plants were grown in a plant growth
chamber for 2 weeks under controlled temperature
(24 ± 2 ◦ C), relative humidity (80%) and light con-
ditions (40 µmol m−2 s−1 , 16-h photoperiod) before
transfer to the greenhouse.
Histological and cytological studies
Anthers were sampled at 7-day intervals from the
inception of cultures and fixed in FAA (formalin/
glacial acetic acid/70% ethanol, 10:5:85) for 12-h at
room temperature, dehydrated in ethanol–butyl al-
cohol series and embedded in paraffin wax (Fowke
and Rennie, 1996). The tissues sectioned at a thick-
ness of 7 µm were stained with 0.05% toluidine-
blue and examined under a compound microscope
(Nikon, Japan). To determine the ploidy level of
the plants, we randomly selected root tips of 22
regenerants of each cultivar and treated them with
2 mM aqueous 8-hydroxyquinoline at room temper-
ature for 4-h. The pretreated roots were fixed in
ethanol:glacial acetic acid (3:1) for 24-h, subse-
quently root tips were washed in water and hydro-
lyzed in 1 N HCl at 60 ◦ C for 10 min. Hydrolyzed
root tips were stained with Feulgen reagent for
1-h and squashed in 45% acetic acid (Armstrong,
1996).
Data collection and analysis
For embryo induction, 60 anthers (12 replicated test
tubes/Petri dishes/conical flasks of five anthers per
genotype) were cultured in each treatment. For plantlet
regeneration, 30 mature embryos were cultured and
all the experiments were repeated three times. The
cultures were observed periodically and morpholog-
ical changes were recorded at weekly intervals. The
number of embryogenic anthers, embryos and plant-
lets produced in each treatment were counted and the
results expressed as percentage of embryogenic an-
thers, number of embryos and plantlets per treatment.
The number of embryos (per 60 anthers) induced and
plantlets (per 30 embryos) regenerated per experiment
were statistically analyzed by analysis of variance
(ANOVA) and mean values were separated according
to Duncan’s multiple range test.
Results and discussion
Effect of sugars on embryo induction
The effects of various concentrations of sucrose, glu-
cose and fructose on embryo induction from cultured
anthers are presented in Table 1. Calypso exhib-
ited direct as well as callus-mediated embryogenesis
while Green Long showed only callus-mediated em-
bryogenesis. During induction of direct embryogen-
esis, anthers swell in 2–3 weeks (Figure 1B) and
induced globular embryos in another 3–4 weeks (Fig-
ure 1D). Histology of 3-week-old anthers showed
enlarged microspores with pro-embryo-like structures
(Figure 1C). During callus-mediated embryogenesis,
anthers become swollen in 2–3 weeks and induced
callus in another 2 weeks (Figure 1F). Putative globu-
lar embryos were formed from embryogenic callus in
another 2 weeks.
Anthers formed embryos on medium containing
sucrose, glucose or fructose. Anthers did not re-
spond on medium devoid of sugar or with only 0.05 M
sucrose, glucose or fructose (Table 1) and also in
all the concentrations of maltose (data not shown).
ANOVA revealed significant difference among the
type and concentration of sugars on embryo induc-
tion. Interestingly, a lower concentration (0.10 M)
of sucrose induced callus-mediated embryogenesis
while higher concentrations of sucrose, glucose and
fructose exhibited direct as well as callus-mediated
embryogenesis in Calypso. In Green Long, only
callus-mediated embryogenesis was observed. In both
cultivars, the percent of responding anthers as well as
number of embryos per treatment increased as the con-
centration of sucrose increased from 0.15 to 0.25 M
(Table 1). The highest numbers of embryos (72 and
80) were induced from 46 and 48% responding anthers
on medium containing 0.25 M sucrose in 6–7 weeks in
Calypso and Green Long, respectively.
Anthers cultured on medium containing 0.1 and
0.15 M glucose or fructose induced only calli in both
the cultivars. In Calypso, 0.2 M glucose and fructose
induced callus. However, in Green Long 0.2 M glu-
cose and fructose induced embryos (Table 1). In
 205
Table 1. Effect of sugars on embryogenesis and plantlet regeneration from cultured anthers of C. sativus L. cvs.
Calypso and Green Long

Sugar M Embryogenesisa,b Plantlet regenerationa,c

Calypso Green Long Calypso Green Long

Responding No. of Responding No. of

anthers (%) embryos anthers (%) embryos

Control – 0 0o 0 0o – –

Sucrose 0.05 0 0o 0 0o – –
0.1 14 5 mn 14 0o 4j –
0.15 34 23 fg 36 31 gh 9 ef 9j
0.2 39 49 b 46 58 b 14 b 15 bc
0.25 46 72 a 48 80 a 18 a 19 a
0.3 37 40 c 42 53 c 15 b 19 a
0.35 31 29 e 33 35 f 15 b 15 b
0.4 23 14 j 21 21 jk 12 c 14 c
0.45 14 8l 16 14 l 10 d 13 d
0.5 6 3n 5 4n 8f 10 hi

Glucose 0.05 0 0o 0 0o – –
0.1 19 0o 12 0o – –
0.15 43 0o 48 0o – –
0.2 39 0o 43 11 l – 4m
0.25 38 14 jk 39 22 j 5i 5 lm
0.3 29 23 fg 36 27 i 7g 6l
0.35 27 24 f 34 33 fgh 9 de 10 ij
0.4 26 30 e 32 41 e 12 c 12 e
0.45 25 36 d 30 49 d 13 c 12 ef
0.5 19 30 e 26 43 e 12 c 11 fg

Fructose 0.05 0 0o 0 0o – –
0.1 17 0o 7 0o – –
0.15 38 0o 42 0o – –
0.2 31 0o 39 7m – 4m
0.25 37 12 k 34 18 k 3k 5 lm
0.3 26 17 i 29 22 j 4j 5l
0.35 22 20 h 29 30 hi 6h 8k
0.4 18 22 gh 26 34 fg 8f 11 gh
0.45 19 30 e 25 35 f 10 d 10 hi
0.5 8 6 lm 8 6 mn 5 ij 3n
a In each column, mean values followed by same letters are not significantly different according to DMRT at
p = 0.05.
b 60 anthers were cultured per treatment.
c 30 mature embryos per treatment were cultured on B5 medium containing 0.09 M sucrose.
Control: B5 + 2.0 µM 2,4-D + 1.0 µM BA and devoid of sugar.

Calypso and Green Long the embryo induction rate
gradually increased as the concentration of glucose
or fructose increased up to 0.45 M (Table 1). In the
present study, sucrose was superior for induction of
embryos to glucose, fructose or maltose and it could
be due to specific carbon effect. Similarly, sucrose has
also been a more suitable carbohydrate source for an-
drogenesis in Brassica oleracea (Yang et al., 1992).
Guo and Pulli (2000) reported that in S. cereale, the
number of embryos/calli per dish were decreased from
116 to 90 as the concentration of sucrose increased
from 0.18 to 0.44 M. Similarly, in the present study,
206
Table 2. Effect of amino acids on embryogenesis and plantlet regeneration from cultured anthers of C. sativus L.
cvs. Calypso and Green Long

Amino acid mM Embryogenesisa,b Plantlet regenerationa,c

Calypso Green Long Calypso Green Long

Responding No. of Responding No. of

anthers (%) embryos anthers (%) embryos

Control 0.0 46 70 fgh 53 81 ghi 17 e 18 cd

Glutamine 0.5 46 71 ef 53 81 fg 18 de 18 cd
1.0 47 72 de 54 83 e 18 de 19 cd
2.0 48 75 b 55 85 c 19 c 20 c
5.0 46 70 fgh 53 81 fg 19 c 20 c

Glycine 0.5 46 70 fgh 53 81 fg 17 e 18 cd

1.0 47 71 ef 54 82 ef 17 e 19 cd
2.0 48 74 bc 55 85 cd 19 c 20 c
5.0 46 71 fg 53 81 ghi 18 de 19 cd

Arginine 0.5 46 70 fgh 53 81 gh 17 e 18 cd

1.0 47 72 de 53 81 fg 18 de 18 cd
2.0 48 74 bc 54 84 cd 19 cd 19 cd
5.0 46 70 fgh 53 80 ghi 18 de 19 cd

Asparagine 0.5 46 71 fg 53 81 ghi 17 e 18 d

1.0 46 71 ef 53 81 fg 18 de 18 cd
2.0 47 73 cd 54 84 d 19 cd 19 cd
5.0 46 70 gh 53 80 hi 18 de 19 cd

Cysteine 0.5 46 71 ef 53 80 ghi 17 e 18 d

1.0 47 72 de 54 82 ef 18 de 18 cd
2.0 47 74 c 54 84 cd 19 cd 18 cd
5.0 46 69 h 53 80 i 18 de 19 cd

Combination 1.0 each 49 79 a 56 91 a 27 a 27 a

Combination 2.0 each 48 74 bc 55 86 b 24 b 25 b

a In each column, mean values followed by same letters are not significantly different according to DMRT at
p = 0.05.
b 60 anthers were cultured per treatment.
c 30 mature embryos per treatment were cultured on B5 medium containing 0.09 M sucrose.
Control: B5 + 2.0 µM 2,4-D + 1.0 µM BA + 0.25 M sucrose.

the efficiency of embryogenesis was significantly re-
duced as the concentration of sucrose increased from
0.25 to 0.50 M in the induction medium (Table 1).
On the contrary, embryo induction increased as the
concentration of glucose or fructose increased from
0.25 to 0.45 M. Embryo induction was reduced when
the concentration of glucose or fructose was further
increased to 0.5 M (Table 1). In earlier work on an-
drogenesis of cucumber (Ashok Kumar et al., 2003),
we used 0.25 M sucrose in embryo induction medium
and confirmed the superiority of this treatment over
a wide range of sugar concentrations in the present
investigation.
Effect of amino acids on embryo induction
The results of the effect of amino acids on anther
culture response in cucumber are summarized in
Table 2. Medium supplemented with glutamine, gly-
cine, arginine, asparagine and cysteine individually
at 2.0 mM showed enhanced embryo induction in
cultured anthers. The same amino acids at 1.0 mM

occasionally enhanced embryo induction. However,
lower a concentration (0.5 mM) as well as a higher
concentration (5.0 mM) did not improve the embryo
yield when compared to control (Table 2). In both
cultivars, among five amino acids tested, glutamine
at 2.0 mM induced the greatest number of embryos
followed by glycine and arginine, cysteine and aspar-
agine (Table 2). In Calypso as well as Green Long, a
combination of amino acids (glutamine, glycine, ar-
ginine, asparagine and cysteine) proved to be best at
1.0 mM (Table 2). Addition of a combination of amino
acids has also been beneficial to induction of embryos
and green plant regeneration in T. aestivum (Trottier
et al., 1993) and H. vulgare (Ouédraogo et al., 1998).
Differentiation, maturation and germination of
embryos
Globular embryos differentiated into cotyledonary
stage embryos on differentiation medium containing
NAA and KN in 4 weeks. The different developmental
stages of embryos were observed in direct as well as
callus-mediated embryogenesis from cultured anthers
of cucumber. Globular (Figure 1D), torpedo (Fig-
ure 1E) and cotyledonary stages (Figure 1G) appeared
successively but heart stage was rare. In cucum-
ber androgenesis, ABA treatment was necessary for
maturation and subsequent development of embryos
into normal plantlets (Ashok Kumar et al., 2003).
Since ABA promotes normal development and pre-
vents precocious germination of zygotic embryos, its
exogenous application may improve normal plant-
let development in androgenesis (Palmer and Keller,
1997). In present study, cotyledonary stage embryos
were matured on maturation medium containing ABA
in 2 weeks. Mature embryos developed into plant-
lets (Figure 1H) on germination medium containing
0.09 M sucrose in another 2 weeks. Among differ-
ent sugars, sucrose exhibited highest frequency of
plantlet regeneration followed by glucose and fructose
(Table 1). Metwally et al. (1998) reported that, induc-
tion medium containing 0.44 M sucrose resulted in the
greatest frequency of plantlet regeneration from cul-
tured anthers of C. pepo. In present study, induction
medium containing 0.25 M sucrose showed maximum
number of plantlet regeneration compared to other
concentrations (Table 1). Addition of alanine, aspar-
agine and glutamine to embryo induction medium
also increased the frequency of embryo differenti-
ation and the percentage of green plant regeneration
in H. vulgare (Muyuan et al., 1990). Similarly, in
207
present study, embryos that developed on medium
containing a combination of amino acids (glutamine,
glycine, arginine, asparagine and cysteine at 1.0 mM
each) showed the greatest frequency of conversion into
plantlets (Table 2).
Hardening of plantlets for 4 weeks under con-
trolled environmental conditions was essential. Of 70
plantlets transferred per cultivar, 31 and 35 plantlets
of Calypso and Green Long survived. Randomly se-
lected regenerants were used for cytological analysis.
In Calypso, 17 of 22 plantlets showed the haploid
chromosome number (n = 7) and the remaining five
plantlets were diploid whereas in Green Long, 14 of
22 regenerants were haploid and the remaining eight
were diploid.
In conclusion, sucrose was the most effective car-
bon source for embryogenesis and plantlet regener-
ation from cultured anthers of cucumber. Addition
of a combination of amino acids to induction me-
dium enhanced both embryo induction and plantlet
regeneration.
Acknowledgements
The authors would like to thank Mr Pramod Menon,
Manager, Agri business, Ken Agritech, Hubli, India
and Manager, Mahyco, Jalna, India for providing
seeds of Calypso and Green Long, respectively. This
work was partly supported by Department of Biotech-
nology (Project No. BT/PR/3153/AGR/16/254/2002),
New Delhi, India.
References
Afza R, Shen M, Zapata FJ, Xie J, Fundi HK, Lee KS, Bobadilla-
Mucino & Kodym A (2000) Effect of spikelet position on rice
anther culture efficiency. Plant Sci. 153: 155–159
Anonymous (1999) Year Book Production 53 (pp. 142–143). FAO,
UN, Rome
Armstrong KC (1996) Staining procedures for chromosome anal-
ysis. In: Gamborg OL & Phillips GC (eds) Plant Cell, Tissue
and Organ Culture. Fundamental Methods (pp. 251–266). Narosa
Publishing House, New Delhi
Ashok Kumar HG, Murthy HN & Paek KY (2003) Embryogenesis
and plant regeneration from anther cultures of Cucumis sativus
L. Sci. Hort. 98: 213–222
Bajaj YPS (1990) In vitro production of haploids and their use in
cell genetics and plant breeding. In: Bajaj YPS (ed) Haploids in
Crop Improvement 1. Biotechnology in Agriculture and Forestry
(pp. 3–44). Springer, Berlin
Bueno MA, Gómez A, Boscaiu M, Manzanera JA & Vicente O
(1997) Stress-induced formation of haploid plants through an-
ther culture in cork oak (Quercus suber). Physiol. Plant. 99:
335–341
208
Chu CC, Hill RD & Brule-Babel AL (1990) High frequency of
pollen embryoid formation and plant regeneration in Triticum
aestivum L. on monosaccharide containing media. Plant Sci. 66:
255
Ferrie AMR, Palmer CE & Keller WA (1995) Haploid embryo-
genesis. In: Thorpe TA (ed) In Vitro Embryogenesis in Plants
(pp. 309–344). Kluwer Academic Publishers, Dordrecht, The
Netherlands
Fowke LC & Rennie PJ (1996) Botanical microtechnique for plant
cultures. In: Gamborg OL & Phillips GC (eds) Plant Cell, Tissue
and Organ Culture. Fundamental Methods (pp. 217–228). Narosa
Publishing House, New Delhi
Gamborg OL, Miller RA & Ojima K (1968) Nutrient requirements
of suspension cultures of soybean root cells. Exp. Cell Res. 50:
151–158
Gémes Juhász A, Venczel G & Balogh P (1996) Haploid plant
induction in zucchini (Cucurbita pepo L. convar. Giromontiina
Duch) and in cucumber (Cucumis sativus L.) lines through in
vitro gynogenesis. Acta Hort. 447: 623–625
Gémes Juhász A, Balogh P, Ferenczy A & Kristóf Z (2002) Effect
of optimal stage of female gametophyte and heat treatment on in
vitro gynogenesis induction in cucumber (Cucumis sativus L.).
Plant Cell Rep. 21: 105–111
Guo YD & Pulli S (2000) Isolated microspore culture and plant
regeneration in rye (Secale cereale L.). Plant Cell Rep. 19:
875–880
Guzmán M & Zapata FJ (2000) Increasing anther culture efficiency
in rice (Oryza sativa L.) using anthers from ratooned plants. Plant
Sci. 151: 107–114
Immonen S & Anttila H (1998) Impact of microspore developmental
stage on induction and plant regeneration in rye anther culture.
Plant Sci. 139: 213–222
Indrianto A, Heberle-Bors E & Touraev A (1999) Assessment of
various stresses and carbohydrates for their effect on the induc-
tion of embryogenesis in isolated wheat microspores. Plant Sci.
143: 71–79
Kiviharju E & Pehu E (1998) The effect of cold and heat pretreat-
ments on anther culture response of Avena sativa and A. sterilis.
Plant Cell Tiss. Org. Cult. 54: 97–104
Lazarte JE & Sasser CC (1982) Asexual embryogenesis and plantlet
development in anther culture of Cucumis sativus L. HortScience
17: 88
Malepszy S (1988) Cucumber (Cucumis sativus L.). In: Bajaj YPS
(ed) Biotechnology in Agriculture and Forestry Vol. 6, Crops II
(pp. 277–293). Springer, Berlin
Metwally EI, Moustafa SA, EI-Sawy BI & Shalaby TA (1998) Hap-
loid plantlets derived by anther culture of Cucurbita pepo. Plant
Cell Tiss. Org. Cult. 52: 171–176
Murthy BNS, Singh RP & Saxena PK (1996) Induction of
high-frequency somatic embryogenesis in geranium (Pelar-
gonium × hortorum Bailey cv. Ringo Rose) cotyledonary cul-
tures. Plant Cell Rep. 15: 423–426
Muyuan Z, Abing X, Miaobao Y, Chunnong H, Zhilong Y, Linji
W & Jianjun Y (1990) Effects of amino acids on callus differ-
entiation in barley anther culture. Plant Cell Tiss. Org. Cult. 22:
201–204
Niemirowicz-Szczytt K & Dumas de Vaulx R (1989) Preliminary
data on haploid cucumber (Cucumis sativus L.) induction. Rep.
Cucurbit Genet. Coop. 12: 24–25
Niimi Y, Han D-S & Fujisaki M (2001) Production of virus-free
plantlets by anther culture of Lilium × ‘Enchantment’. Sci. Hort.
90: 325–334
Ouédraogo JT, St-Pierre C-A, Collin J, Rioux S & Comeau A
(1998) Effect of amino acids, growth regulators and geno-
type on androgenesis in barley. Plant Cell Tiss. Org. Cult. 53:
59–66
Owen HR & Miller AR (1996) Haploid plant regeneration from
anther cultures of three North American cultivars of strawberry
(Fragaria × ananassa Duch.). Plant Cell Rep. 15: 905–909
Palmer CE & Keller WA (1997) Pollen embryos. In: Shivanna KR
& Sawhney VK (eds) Pollen Biotechnology for Crop Production
and Improvement (pp. 392–422). Cambridge University Press,
Cambridge, UK
Przyborowski J & Niemirowicz-Szczytt K (1994) Main factors
affecting cucumber (Cucumis sativus L.) haploid embryo de-
velopment and haploid plant characteristics. Plant Breed. 112:
70–75
Rizza F, Mennella G, Collonnier C, Sihachakr D, Kashyap V, Rajam
MV, Prestera M & Rotino GL (2002) Androgenic dihaploids
from somatic hybrids between Solanum melongena and S. ae-
thiopicum group gilo as a source of resistance to Fusarium
oxysporum f. sp. Melongenae. Plant Cell Rep. 20: 1022–1032
Sauton A (1989) Haploid gynogenesis in Cucumis sativus induced
by irradiated pollen. Rep. Cucurbit Genet. Coop. 12: 22–23
Trottier M-C, Collin J & Comeau A (1993) Comparison of media
for their aptitude in wheat anther culture. Plant Cell Tiss. Org.
Cult. 35: 59–67
Yang Q, Chauvin JE & Hervé Y (1992) A study of factors affecting
anther culture of cauliflower (Brassica oleracea var. botrytis).
Plant Cell Tiss. Org. Cult. 28: 289–296