Plant Cell Tiss Organ Cult (2012) 109:167–170
DOI 10.1007/s11240-011-0066-9
RESEARCH NOTE
Parthenogenetic haploid plants using gamma irradiated pollen
in snapmelon (Cucumis melo var. momordica)
Manoj Godbole • Hosakatte Niranjana Murthy
Received: 1 August 2011 / Accepted: 24 September 2011 / Published online: 2 October 2011
Ó Springer Science+Business Media B.V. 2011
Abstract Anthers of snapmelon (Cucumis melo L. var.
momordica) were irradiated with varied doses of gamma
rays (150, 200, 250, 300 and 350 Gray). Then pollen from
irradiated anthers was used for pollinating female flowers.
Results revealed that 250 Gray of gamma-irradiation was
successful in inducing parthenogenesis and fruit develop-
ment, whereas, low (150 and 200 Gray) or high (300 and
350 Gray) irradiation doses were not effective in inducing
haploid embryos. Embryos at a range of developmental
stages were dissected from fruits harvested after 21 days of
pollination and cultured on E20A medium. Among these
embryos cultured, only cotyledonary embryos germinated
into plantlets. Chromosome counting, performed on the
roots of regenerated plants, showed the haploid level
(n = 12). Ploidy analysis using flow cytometer, measure-
ment of stomatal cells and counting of chloroplast in the
guard cells also corroborated the haploid nature of regen-
erated plants.
Keywords Cucumis melo var. momordica
Gamma
irradiation
Snapmelon
Haploid
Embryo rescue

Parthenogenesis
Snapmelon (C. melo var. momordica, Cucurbitaceae) is an
important fruit crop in India where it is commonly known
as ‘phut’ which means to split. Immature fruits of snap-
melon are cooked or pickled. The mature fruits with low
sugar content are eaten raw and when ripe, the fruits
invariably crack. Snapmelon germplasm has been found to
M. Godbole
H. N. Murthy (&)
Department of Botany, Karnatak University, Dharwad,
Karnataka 580 003, India
e-mail: nmurthy60@yahoo.co.in
be a very good source of disease and insect resistance
(Dhillon et al. 2007). Powdery mildew, downy mildew,
fusarium wilt, zucchini yellow mosaic virus (ZYMV),
papaya ringspot virus (PRSV), cucurbit aphid borne yellow
virus (CABYV) and aphis gossypii glover resistant snap-
melon are being cultivated in various parts of India (Pitrat
et al. 2000). A major goal of melon breeding is to develop
resistance to the many fungal and viral diseases that affect
this crop (Anagnostou et al. 2000).
Haploids and doubled haploids play an important role in
plant breeding by shortening the breeding time required to
produce the homozygous plants. Homozygous lines are
often considered as a first step in genetic improvement of
vegetable crops and they can be used for production of
hybrids, synthetic cultivars and new cultivars directly.
Haploids can be obtained via the routes of androgenesis,
gynogenesis or parthenogenesis (Germana 2011; Chen
et al. 2011). The first reported success in obtaining haploid/
doubled haploid muskmelon was achieved by rescue of
parthenogenetic embryos induced by pollination with
irradiated pollen (Sauton and Dumas de vaulx 1987). This
was confirmed and investigated further by other authors
working with cucurbits (Lotfi et al. 2003; Sari et al. 1994;
Kurtar et al. 2002). There are no reports on induction of
haploids in snapmelon, so, the aim of this research was to
investigate the possibility of inducing haploid embryos of
C. melo var. momordica through the irradiated pollen
technique.
Seeds of snapmelon (Cucumis melo var. momordica)
were sown in pots with a mixture of 1:1:1 sand, soil and
farm yard manure. Plants were watered on every alternative
day and commercial fertilizers were supplied every fort-
night and plants were protected with insecticides and fun-
gicides regularly throughout the growing season. Male
flower buds were collected 1 day before anthesis: they
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were placed in Petri-dishes after removal of the petals.
Then they were gamma irradiated at 150, 200, 250, 300 and
350 Gy using a 60Co source (M.C.M Phoenix, Germany)
with the output of 6.4683 Gy/min at the Karnataka Institute
of Medical Sciences, Hubballi and then stored overnight at
room temperature. The female flower buds were covered
with bags made up of muslin cloth to prevent any chances
of pollination. The following day, female flowers were
pollinated with pollen collected from three irradiated male
flowers and covered again with a muslin cloth bag.
Well developed fruits were harvested after 21 days. The
fruits were washed with running tap water followed by
Tween 20 for 5 min, and then they were sprayed with 70%
alcohol. Further surface sterilization was carried out in
aseptic condition with 0.1% mercuric chloride for 15 min
and rinsed with sterile distilled water three times. The
sterilized fruits were dissected; each seed was opened and
checked for the development of the embryo. The embryos
thus obtained were cultured on E20A medium (Sauton and
Dumas de vaulx 1987) supplemented with 2% sucrose and
0.06 lM indole-3-acetic acid (IAA). The cultures were
incubated under a 16 h photoperiod of 40 lmol m-2 s-1
and at a temperature of 25 ± 2°C. Plantlets obtained after
embryo germination were multiplied in vitro by nodal
cuttings on the E20A medium (Sauton and Dumas de vaulx
1987) supplemented with 2% sucrose and 0.06 lM IAA.
After 2 weeks of culture, embryos germinated and devel-
oped into plantlets. Well developed plantlets (15 cm in
height) were taken out of the culture vessel and washed
thoroughly in sterile distilled water and transferred to pots
containing a mixture of autoclaved vermiculite, sand and
soil in the ratio 1:1:1 and were maintained in a growth
chamber (Sanyo, Osaka, Japan) at 25°C, 60% relative
humidity, with a 16 h photoperiod (40 lmol m-2 s-1)
provided by 40-W fluorescent lamps. After hardening for
4 weeks the plantlets were transferred to pots containing
sand, soil and farmyard manure in the ratio 1:1:1 and raised
in the green house.
Ploidy analysis of regenerated plants was determined by
using PA-I ploidy analyzer (Partec, Munster, Germany)
according to the protocol of Kiełkowska and Adamus
(2010). Chromosome counts have been also carried out by
staining root tips in 2% aceto-orcein as per the method
described by Doi et al. (2010) and Winarto et al. (2011).
Stomata dimensions and chloroplast number of the guard
cells were determined as per the protocol of Rego et al.
(2011).
Anthers of snapmelon were irradiated with varied doses
of c-rays (150, 200, 250, 300 and 350 Gray) and subse-
quently pollen from irradiated anthers was used for polli-
nation of female flowers. Results revealed that 250 Gray of
c-irradiation was successful in inducing parthenogenesis
and fruit development (Table 1, Fig. 1a). Lower (150 and
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Plant Cell Tiss Organ Cult (2012) 109:167–170
Table 1 Effect of c-irradiation of pollen from male flower on fruit
setting after pollination of female flowers in Cucumis melo var.
momordica
Irradiation Fruit Seeds Seed with Plantlet
dose (Gy) setting cultured embryo developed
150 0 – – –
200 0 – – –
250 3 708 119 2
300 0 – – –
350 0 – – –
200 Gray) or higher (300 and 350 Gray) irradiation doses
were not effective on embryo induction and gave fruits
which were seedless. It was reported that embryo and
haploid plants were obtained from lower irradiation doses
in summer squash (25 and 50 Gray; Kurtar et al. 2002) and
pumpkin (50 and 100 Gray; Kurtar and Balkaya 2010). On
the other-hand, a higher dose of irradiation was necessary
in watermelon (200–300 Gray; Gursoz et al. 1991; Sari
et al. 1994), melon (Lotfi et al. 2003) and cucumber (Ca-
glar and Abak 1999). The present study reveals that a
moderate dose of 250 Gray was effective in parthenoge-
netic embryo induction in snapmelon. It appears that
induction of parthenogenesis via irradiated pollen is spe-
cies specific and the irradiation dose levels must be worked
out in the experimental species.
Embryos of different developmental stages viz. globu-
lar, heart, cotyledonary shaped embryos and even irregular
embryos were rescued and classified according to the
stages of development (Raghavan 1986; Kurtar et al. 2002;
Table 2, Fig. 1b). Overall 119 embryos were rescued, out
of them the majority of embryos were globular (83.19%)
and heart staged (5.04%) which did not show further
development and plant regeneration. Even though fourteen
cotyledonary (11.76%) embryos were rescued only two
was regenerated into plants (Fig. 1c). Regenerated plants
were multiplied in vitro thorough nodal cuttings. The
regeneration rate was reportedly influenced by develop-
mental stage of the embryos and cotyledon embryos had
the highest regeneration rate in pumpkin (66.75%; Kurtar
and Balkaya 2010) and cucumber (81.8%; Caglar and Abak
1999). Thus, success of embryo rescue depends on the
stage of embryo development and culture media as repor-
ted by Jaskani et al. (2005).
Regenerated plants were hardened and maintained in the
green house (Fig. 1d). Haploid plants were less vigorous and
had smaller leaves and flowers with a few empty pollen grains
when compared to diploid plants. Although our results indi-
cated a low frequency of haploid plantlets (1.68%), this is the
first report on induction of parthenogenetic haploid embryo
via irradiated pollen technique in snapmelon. Low frequency
haploid may be explained by the greatest number of immature
Plant Cell Tiss Organ Cult (2012) 109:167–170 169

Fig. 1 Induction of haploid

plants after pollination by
irradiated pollen in Cucumis
melo var. momordica.
a Parthenogenetic fruit
development after pollination
with pollen irradiated with 250
Grays of gamma rays
(bar = 36 mm).
b Cotyledonary embryo
dissected from the
parthenogenetic seed
(bar = 1.1 mm). c Germination
of the cotyledonary embryo on
E20A medium supplemented
with 2% sucrose and 0.06 lM
IAA (bar = 1.7 mm).
d Acclimatized parthenogenetic
plant (left) compared to the
control plant (right)
(bar = 89.4 mm). e Squash
preparation of root tips showing
12 chromosomes (n = 12)
(bar = 0.024 mm). f Flow
cytometric analysis showing the
haploid nature of the plantlet.
g Stomatal guard cell of leaf of
haploid plant having 4
chloroplasts (bar = 0.024 mm).
h Stomatal guard cell of leaf of
diploid plant having 12
chloroplasts (bar = 0.024 mm)

Table 2 Different staged embryos formed after the pollination of

embryos (globular and heart shaped), and the subsequent
female flowers with c-irradiated pollen in Cucumis melo var. mo- absence of regeneration. Similar to the present results, a low
mordica and their conversion into plantlets on E20A medium sup- frequency of haploid plant regeneration via parthenogenesis
plemented with 2% sucrose and 0.06 lM IAA has been reported in melon (Lofti et al. 2003). Counting of
Embryo stage Number Percentage Plantlet chromosomes in roots of regenerated plants, showed the
of embryos formed haploid chromosome number (n = 12; Fig. 1e). Flow cyto-
Globular 99 83.19 0
metric analysis revealed that the regenerated plants were
haploid in nature containing 1C DNA level when compared to
Heart 06 5.04 0
diploids which had 2C DNA (Fig. 1f). Stomatal length,
Cotyledonary 14 11.76 2
diameter and chloroplast number in guard cells differed

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Table 3 Guard cell stomata
Stomatal length (lM)
characteristics of haploid and
diploid plants of Cucumis melo Haploid Diploid
var. momordica
16.48 24.72
16.48 24.72
14.36 24.72
14.36 20.6
16.48 24.72
14.36 20.6
14.36 20.6
14.36 24.72
between haploid and diploid plants. The average stomatal
length 9 diameter was 15.15 9 12.3 lm in haploid plants,
while in control plants stomatal length and diameter was
23.17 9 15.69 lm. Average chloroplast numbers in the
guard cells of haploid plants were 4.37 and 12.25 chloroplasts
were recorded in diploid plants (Table 3, Fig. 1 g, h). Sari
et al. (1994) in watermelon, Rode and Dumas de vaulx (1987)
in carrot found similar results. Thus, the stomatal size and
chloroplast number of guard cells could be alternative criteria
to determine the ploidy level in haploid/diploid plants.
In conclusion, our results indicated that pollination by
gamma irradiated pollen can induce haploid embryo
development in snapmelon and this study is the first suc-
cessful report in this species. However, the frequency of
haploid embryo induction was very low, further research
should be focused on improvement of this method for large
scale production of haploid plants.
Acknowledgments Authors thank the University Grants Commis-
sion, New Delhi, India for the financial assistance [F. No. 33–188/
2007 (SR)], Director, Karnataka Institute of Medical Sciences,
Hubballi, India for the radiation facility and Directorate of Oilseed
Research, University of Agricultural University Campus, Hyderabad
for sparing the flow cytometer facility.
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