Plant Cell, Tissue and Organ Culture 52: 117–121, 1998.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

117

Production of haploid plants from in vitro culture of unpollinated ovules of

Cucurbita pepo

E.I. Metwally, S.A. Moustafa, B.I. El-Sawy, S.A. Haroun1 & T.A. Shalaby
Horticulture Department, Faculty of Agriculture, Kafr El-Sheikh, 33516, Egypt. 1 Biology Department, Faculty of
Education, Kafr El-Sheikh, 33516, Egypt

Received 13 November 1996; accepted in revised form 8 January 1998

Key words: Cucurbita pepo L., ovule culture

Abstract
Ovaries from squash plants (cv. Eskandarani) were picked one day before anthesis, and exposed to cold temperature
(4 ◦ C) for 0, 2, 4 and 8 days. The ovules were cultured on MS medium with 30 g l−1 sucrose, 8 g l−1 agar and
supplemented with four concentrations of 2,4-D, i.e., 0.1, 1.0, 5 and 10 mg l−1 . Then the dishes were incubated
at 25 ± 1 ◦ C under 16-h photoperiod for 4 weeks. After that ovules were transferred to growth regulator free
MS medium for 4 weeks. Data indicated that the most plantlets per 100 cultured ovules resulted from the ovule
of ovaries without cold treatment, when cultured in MS medium supplemented with 1 or 5 mg l−1 2,4-D. The
cytological study revealed that one third of examined plants were haploid (2n = x = 20) and the others were double
haploid (2n = 2x = 40).

Introduction the years 1993 to 1995. Seeds of squash, Cucurbita

Pure lines are often considered a first step in genetic
improvement of vegetable crops. Pure lines can be
used for three purposes, i.e., production of superior
hybrids, synthetic cultivars and new cultivars directly.
The production of pure lines in different crops, espe-
cially open-pollinated plants like Cucurbita pepo L.,
requires both time and adequate facilities. Recently,
pure lines (doubled haploid plants) can be obtained
in a short time by using in vitro anther and/or ovule
culture. However, anther culture fails in some cases
such as female plants and male sterile plants (Van
Geyt et al., 1987 and Doctrinal et al., 1989). When
morphogenesis occurs in the gametophytic cells, it is
remarkably efficient for the rapid production of ho-
mozygous lines and the detection of mutation. Early
work showed such fact (Pallares, 1984). So, the aim
of this research was to improve the efficiency of ovule
culture technique for the production of haploid plants
in Cucurbita pepo L. through in vitro gynogensis.
Materials and methods
This work was carried out at the Horticulture Depart-
ment, Faculty of Agriculture at Kafr El-Sheikh during
pepo L. cv. Eskandarani, were sown in the Experi-
mental Farm of the Faculty during the summer season
of 1993 in order to obtain female flower buds for the
laboratory work.
This experiment was conducted to study the ef-
fect of temperature pretreatments (at 4 ◦ C for 0, 2,
4 and 8 days) and different concentrations of 2,4-
dichlorophenoxyacetic acid (2,4-D) (0.1, 1.0, 5.0 and
10 mg l−1 ) and their interaction on induction of hap-
loid plants through unfertilized ovule culture. A split
plot design with four replications was used. Temper-
ature pretreatments were arranged in the main plots
and four concentrations of 2,4-D were assigned in the
subplots. Therefore MS medium supplemented with
30 g l−1 sucrose, 8 g l−1 agar and four concentrations
of 2,4-D were used. The pH of media was adjusted to
5.7 and the media were autoclaved at 121 ◦ C for 15
min. The experiment included 16 treatments.
Ovaries were harvested one day before anthesis as
recommended by Dumas de Vaulx and Chambonnet
(1986) and exposed to cold temperature. After the cold
pretreatments, petals and style were removed. Then
the ovaries were sterilized by soaking in 70% ethanol
for 3 min and rinsed three times with sterile distilled

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Figure 1. Regenerated plantlets from ovule culture
water. They were placed on a sterilized paper towel to
absorb surface water.
The ovules were excised from ovaries in a laminar
flow cabinet and placed in the different induction me-
dia. Each treatment included four replications, each
replicate (dish) containing 100 ovules. The dishes
were sealed with parafilm and incubated at 25 ± 1 ◦ C
under 16-h photoperiod for 4 weeks. Then ovules were
transferred to growth regulator-free MS medium for 4
weeks, and the following data were recorded:
(1) number of plantlets per 100 cultured ovules
(2) number of gynogenic ovules per 100 cultured
ovules
(3) number of calluses per 100 cultured ovules
Data were analyzed using SAS program (SAS, 1989)
on IBM personal computer.
After plantlets were obtained from ovule culture
in petri dishes (Figure 1) they were transplanted indi-
vidually into Magenta boxes containing MS medium
without growth regulators (Figure 2) and kept under
16-h photoperiod (3000 lux) at 25±1 ◦ C for 4 weeks.
Plant acclimatization was as described by Shalaby
(1996).
For mitotic studies, root tips were treated with
0.05% aqueous solution of colchicine for 3 h, then
Figure 2. Plantlet in Magenta box
fixed in normal fixative solution. Normal squash tech-
nique was carried out by Feulgen stain. Photographs
were taken from permanent slides (× 4000 magnifica-
tion).
Results and discussion
Effect of cold temperature pretreatment:
Effect of cold treatment of ovaries on the production of
embryos are presented in Table 1. Ovules from flower
buds without cold treatment produced a better em-
bryogenic response than the ovules from flower buds
treated at 4 ◦ C for 2, 4 or 8 days. The greatest number
of plantlets per 100 cultured ovules resulted from the
ovules of ovaries without cold treatment. Preliminary
work indicated that in unpollinated ovule culture, em-
bryogenesis occurs in the embryo sac cells, especially
in the egg in which an embryo (embryogenic ovules),
then the embryo grows directly may develop into hap-
loid plantlet or gives rise to callus (Pallares, 1984).
Data in Table 1 show that both nontreated flower at
buds or those treated 4◦ C for 8 days gave the mot
calluses per 100 cultured ovules. On the other hand
flower buds treated with 4 ◦ C for 2 days gave the
fewest calli.
ticu2513.tex; 27/04/1998; 11:09; p.2
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Table 1. Effect of cold temperature treatments on induction of em-

bryos in unfertilized ovules of squash.

Period of cold No. of plantlets/ No. of No. of

treatment (days) 100 ovules gynogenic calluses
(4◦ C) ovules/ 100 ovules
100 ovules

0 11.0 a 7.5 a 5.3 a

2 3.9 c 2.0 c 2.1 c
4 4.3 c 2.3 c 2.9 b
8 8.9 b 5.8 b 5.4 a

Means followed by a common letter are not significantly different at

the 5% level according to Duncan’s test.

Table 2. Effect of 2, 4-D concentration on the production of embryos in

unfertilized ovules of squash.

2,4-D No. of plantlets/ No. of gynogenic No. of

concentration 100 ovules ovules/ calluses/
mg l−1 100 ovules 100 ovules

0.1 4.9 b 2.5 c 2.1 c

1.0 8.8 a 5.0 a 3.5 b
5.0 9.1 a 6.5 a 5.3 b
10.0 5.3 b 3.4 b 4.9 a

Means followed by a common letter are not significantly different at the

5% level according to Duncan’s test.

Table 3. Effect of cold treatments-2,4-D interaction on production of

embryos in unfertilized ovules of squash.

Treatment No. of No. of No. of

Cold 4◦ C 2,4-D plantlets/100 gynogenic calluses/100
(days) (mg l−1 ) ovules ovules/100 ovules
ovules

0 0.1 0.5 e 0.5 d 0.5 f

1.0 18.0 a 11.5 a 4.5 bcd
5.0 17.5 a 12.5 a 10.0 a
10.0 8.0 bcd 5.0 bc 6.0 bc
2 0.1 6.0 cd 3.5 cd 1.5 ef
1.0 4.0 de 3.0 cd 2.0 def
5.0 2.0 de 1.5 cd 4.5 bcd
10.0 3.5 de 1.5 cd 4.5 bcd
4 0.1 6.0 cd 3.0 cd 2.5 def
1.0 3.5 de 2.0 cd 2.5 def
5.0 5.5 cd 3.0 cd 4.0 cde
10.0 2.0 de 1.5 cd 2.5 def
8 0.1 7.0 bcd 3.5 cd 4.0 cde
1.0 9.5 bc 5.5 bc 4.0 cde
5.0 11.5 b 9.0 ab 7.0 b
10.0 7.5 bcd 5.0 c 6.5 bc

Means followed by a common letter are not significantly different at the

5% level according to Duncan’s test.

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Figure 3. (a) Mitotic metaphase of haploid plant, 2n = x = 20. (b) Metaphase stage of squash root-tip cell with 40 chromosomes.
Cold treatment at 4◦ C for 2, 4 or 8 days sup-
pressed of embryogenesis compared with the control.
The control ovules (without cold treatment) gave the
highest embryogenic ovules per 100 cultured ovules.
Yang and Zhou (1982) reported that cold temperature
pretreatment was ineffective in ovary and ovule cul-
ture of most species. On the other hand, Kwack and
Fujieda (1988) found that the cold treatment of Cucur-
bita moschata ovaries at 5 ◦ C for 2 days was efficient
for embryogenesis. Lux et al. (1990) found that cold
pretreatment of sugar beet flower buds at 4◦ C led to
increased embryo yield with a maximum at 4-5 days.
Also, Hanna (1994) reported an enhancing effect for
cold pretreatment at 4◦ C for 4 days in onion.
Effect of 2, 4-D concentration:
Data presented in Table 2 show that the number of
plantlets increased with increasing auxin (2,4-D) from
0.1 mg l−1 up to 5.0 mg l−1 . The media supplemented
with 1.0 or 5.0 mg l−1 2,4-D gave the most plantlets
(8.8 and 9.1 per 100 cultured ovules respectively). The
media supplemented with either 0.1 or 10.0 mg l−1
2, 4-D gave the lowest number of plantlets per 100
cultured ovules. The differences between the largest
and the lowest were statistically significant.
Also, the ovules producing embryogenic callus in-
creased with increasing the auxin from 0.1 mg l−1 to
5.0 or 10.0 mg l−1 2,4-D. The difference between the
largest and lowest was significant.
The number of gynogenic ovules per 100 cultured
ovules increased with increasing auxin concentration
up to 5.0 mg l−1 2, 4-D. The media containing 1.0 or
5.0 mg l−1 2, 4-D gave the gynogenic ovules (5.0 and
6.5 per 100 cultured ovules), respectively. The differ-
ences among treatments were significant. According
to Evans et al. (1981), the primary medium for induc-
tion of somatic embryogenesis includes 2,4- D for the
majority of crop species. Kwack and Fujieda (1988)
indicated that plant growth regulators are important for
embryogenesis of Cucurbita moschata. Also, Cam-
pion et al. (1992) found that 2, 4-D gave good results in
both ovary and flower culture of onion. Bridgen (1994)
found that somatic embryo induction was inhibited by
high concentrations of exogenous auxin in the medium
and stimulated by low concentrations.
Effect of cold pretreatment-2,4-D interaction:
The differences among treatments were significant for
number of plantlets per 100 cultured ovules, number
of calluses per 100 cultured ovules, and number of
gynogenic ovules per 100 ovules (Table 3). The most
plantlets per 100 cultured ovules were obtained from
ovaries without previous cold treatment and cultured
on MS medium supplemented with 1.0 or 5.0 mg l−1 .
ticu2513.tex; 27/04/1998; 11:09; p.4

Most calluses per 100 cultured ovules were ob-
tained when the ovaries were not treated with cold
temperature and cultured on MS medium supple-
mented with 5 mg l−1 2,4-D.
Cytological studies
Root-tips from 20 gynogenetic regenerant plants
(plants derived from ovule culture) were cytologically
examined under light microscope. Results (Figure 3A,
B) revealed 15 diploid (2n = 2x = 40) and 5 haploid
(2n = x = 20) plants. Whitaker and Davis (1962) in-
dicated that there is ample evidence that all species of
Cucurbita have twenty pairs of chromosomes. How-
ever, the small size of the mitotic chromosomes in this
genus makes them troublesome to count accurately,
even though they tend to be well separated.Varghese
(1973) reported that little is known about the cytology
of Cucurbita genus because its cytological studies are
difficult, and that all the species have 2n = 40 chromo-
somes. On the other hand Dryanovska (1985) found
that diploid, haploid and aneuploid metaphases were
observed in the callus tissue derived from ovaries and
anthers of species from the Cucurbita family.
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