Plant Cell Tiss Organ Cult (2008) 95:115–124

DOI 10.1007/s11240-008-9422-9

ORIGINAL PAPER

Effect of in vitro and in vivo colchicine treatments on pollen

production and fruit set of melon plants obtained
by pollination with irradiated pollen
Wansang Lim Æ Elizabeth D. Earle

Received: 20 March 2008 / Accepted: 20 June 2008 / Published online: 16 July 2008
Ó Springer Science+Business Media B.V. 2008

Abstract Haploid/doubled haploid (DH) technol-
ogy can aid plant breeding programs by accelerating
production of homozygous lines, provided enough
viable DH progeny can be obtained from diverse
haploid genotypes. In cases where there is a low
frequency of spontaneous doubling, chromosome
doubling procedures are required to achieve fecun-
dity. We produced 63 parthenogenetic melon
plantlets via pollination with c-irradiated pollen,
cloned them by nodal cuttings, and tested the effects
of in vitro and in vivo colchicine treatment on
survival, ploidy, pollen production, and fruit recov-
ery. The most effective procedure was in vitro
exposure of 3 cm shoot tip explants to 500 mg/l
colchicine for 3 h. This treatment gave 83% survival
of explants and 26% conversion to diploidy. Fruit
recovery rate was 60% among plants with good
pollen production. In vivo exposure of the tops of
young plants to 5000 mg/l for 2 and 4 h yielded some
fruits but also resulted in less survival and more
morphological abnormalities. Strategies for recovery
of progeny from parthenogenetic melon plants are
recommended. To our knowledge, this study repre-
sents the first comprehensive study of recovery of
fruits and viable seeds from parthenogenetic melon
plants.
W. Lim
E. D. Earle (&)
Department of Plant Breeding & Genetics,
Cornell University, Ithaca, NY 14853, USA
e-mail: ede3@cornell.edu
Keywords Cucumis melo
Doubled haploid

Flow cytometry
Parthenogenesis
Abbreviations
APM Amiprofos methyl
DH Doubled haploid
DMSO Dimethyl sulfoxide
IAA Indole-acetic acid
Introduction
Doubled haploids (DH) can aid plant breeding
programs by reducing the time required to obtain
homozygous inbred lines. Several in vivo or in vitro
methods have been used to obtain haploids from
important crops. In melon, haploids have been
recovered both by culture of unpollinated ovaries
(Ficcadenti et al. 1999) and from parthenogenetic
embryos produced after pollination with irradiated
pollen (Sauton and Dumas de Vaulx 1987; Sauton
1988; Cuny et al. 1993; Sari and Abak 1994; Doré
et al. 1995; Ficcadenti et al. 1995; Lotfi et al. 2003).
A constraint in the application of haploid technology
to crop improvement is doubling of the haploid
chromosome number to obtain fertile plants. Conver-
sion of melon haploids to diploids has been attempted
using either in vitro or in vivo treatment with
colchicine; however, the most effective protocol for

123
116
chromosome doubling and recovery of fruits and
viable seeds is not yet clear.
The goals of this work were to apply several types
of chromosome doubling techniques to parthenoge-
netic melon obtained through gynogenesis after
pollination with irradiated pollen and to compare
the effects of these treatments on survival, ploidy,
pollen production, and recovery of fruit and viable
seed from the treated materials.
Materials and methods
Plant materials
Three genotypes of melon (Cucumis melo L.) were
used as initial plant materials. The following three
hybrid genotypes were BC4F1 populations from Dr.
Molly Jahn’s melon breeding program at Cornell
University. They segregate for resistance to gummy
stem blight and powdery mildew, as well as zucchini
yellows and papaya ringspot viruses.
510: [[[[ZPPM 9 [511890 9 482399]F3 9 [[ZPPM
9 (MAIN 9 157082)]F2] 9 [ZPPM 9 (140471 9
482398)]F2]F2]F4 9 Oro Rico]F2]BC4F1
518: [[[[ZPPM 9 [511890 9 482399]F3 9 [[ZPPM
9 (MAIN 9 157082)]F2] 9 [ZPPM 9 (140471 9
482398)]F2]F2]F4 9 HMX 0583]F2]]BC4F1
521: [[[[ZPPM 9 [511890 9 482399]F3 9 [[ZPPM
9 (MAIN 9 157082)]F2] 9 [ZPPM 9 (140471 9
482398)]F2]F2]F4 9 Oro Rico]F2]]BC4F1
The numbers in these lineages (e.g., 511890,
482399, etc.) represent the PIs used as sources of
disease resistance. The names ZPPM, MAIN, Oro
Rico, and HMX represent commercial parents in the
crosses.
Recovery of plants from parthenogenetic embryos
The procedure for recovery of plants from partheno-
genetic embryos was based on Lotfi et al. (2003) with
some modifications. In June–July and November–
December of 2005, male flowers were collected in the
afternoon on the day before anthesis. After removal
of petals, the flowers were irradiated with 250 Gray
(25,000 Rad) using a 137Cs source (Gammacel) and
then stored overnight at room temperature in sealed
35 mm plastic Petri dishes. Most female flowers were
123
Plant Cell Tiss Organ Cult (2008) 95:115–124
hermaphrodite and were emasculated when the male
flowers were collected. The stigmas of all female
flowers were covered with a gelatin capsule to
prevent uncontrolled pollination.
The following day, large female flowers were
pollinated with pollen collected from 2 to 3 irradiated
male flowers. Large fruits ([10 cm in diameter) were
harvested about 3 weeks after pollination. Several
smaller fruits were harvested later, after they grew to
normal size (*10 cm). Seeds were removed from the
fruits, washed with running tap water, sterilized for
20 min with 20% Clorox (1.05% NaOCl) containing
1% Tween 20, and then rinsed 3 times with sterile
water. The seeds were cultured in 10 cm 9 20 mm
plastic Petri dishes (*80 seeds/dish) containing 20
ml of liquid E20A medium (Sauton et al. 1989) and
examined periodically over a light box (Fig. 1a).
Seeds that contained embryos were dissected asepti-
cally after 10–15 days of culture in liquid medium,
and the embryos (Fig. 1b) were transferred to Petri
dishes (10 embryos/dish) containing 20 ml of E20A
medium solidified with 0.8% Phytoblend agar (Cais-
son Laboratories, Inc., North Logan, UT). When
embryos had 1–1.5 cm shoots (Fig. 1c) they were
moved to Magenta boxes containing 40 ml of solid
E20A medium. To establish clonal populations, we
repeatedly subcultured by placing 4 or 5 single node
cuttings containing an axillary bud horizontally in
Magenta boxes containing solid E20A medium. All
media were sterilized by autoclaving at 121°C, 18
psi. Cultures were maintained at 25°C, 16 h
photoperiod.
In vitro colchicine treatments
Colchicine (Sigma) was dissolved in dimethyl sulf-
oxide (DMSO) and diluted in distilled water to 10
mg/ml. This colchicine stock was filter sterilized. The
stock was further diluted in sterile distilled water to
final concentrations of 500 and 1000 mg/l.
Rapidly growing in vitro plantlets were stripped
of leaves. Shoot tips (1.5 or 3 cm long) and nodes
(*1.5 cm) were then placed into baby food jars
containing 25 ml colchicine solution for 3 h on a
shaker at 100 RPM. After 3 washes with sterile
water, explants were transferred to solid E20A
medium in Magenta boxes. Plantlets that developed
roots were transferred to 7.5 cm pots with Cornell
soilless mix (Sheldrake and Boodley 1973). Shoots
Plant Cell Tiss Organ Cult (2008) 95:115–124
Fig. 1 (a) Melon seeds
from fruit obtained after
pollination with irradiated
pollen cultured in liquid
E20 medium, with seed
containing an embryo
circled (b) Melon embryo
excised from cultured seed
(c) Melon plantlet derived
from excised embryo (d)
Treatment of melon plants
with colchicine in vivo
without roots were transferred to rooting medium
(E20A medium containing 0.175 mg/l filter-steril-
ized indole-3-acetic acid [IAA]) before potting.
Plants in pots were covered with a plastic sandwich
bag, the corners of which were cut open after 3 and
6 days to allow ventilation. The bag was removed
after 9 days. Fully acclimatized plants were taken
to a greenhouse, eventually transferred to 22.5 cm
pots, and grown to maturity. Greenhouse conditions
were 25°C day and 20°C night, with a 16 h
photoperiod (5 a.m. to 9 p.m.).
In vivo colchicine treatments
Five to 10 cm long haploid and mixoploid plantlets
were transferred from in vitro conditions to 15 cm
pots of Cornell soilless mix and grown to a height
of 15–20 cm. Four to six plants were then placed
around 250 ml jars (Fig. 1d) containing a solution
of colchicine dissolved in DMSO and diluted in
distilled water. The tips and the first leaf from the
top were dipped into either 1000 mg/l colchicine
for 12 h or into 5000 mg/l colchicine for 2 or 4 h.
One week later, the dipped parts were washed 3
times with running tap water, and plants were
transferred to the greenhouse. Two weeks later, the
plants were transferred to 22.5 cm pots for further
growth. The greenhouse conditions were as
described above.
117
Pollinations of culture-derived plants
Greenhouse-grown plants were self pollinated
between 8 and 10 a.m. Pollen from one to three
male flowers was used for each female flower.
Pollinations were continued until each plant either
had fruit or was dead.
Pollen counts
All of the anthers were removed from a male flower
on each greenhouse-grown plant and teased apart
with forceps in a 40 ll drop of Alexander’s stain
(Alexander 1969) on a microscope slide. The stained
pollen grains were covered with an 18 mm cover slip
and observed with an Olympus CK2 inverted micro-
scope (1009). Stained large round pollen grains were
considered to be fertile and viable; unstained shriv-
eled pollen was considered sterile. Normal appearing
pollen occurring in four microscope fields of each
sample was counted and averaged. This average
value is the ‘‘pollen number’’ presented in the text
and tables. For a sample of flowers (*15), the pollen
number was compared with the total number of
viable pollen grains in a whole male flower. All the
anthers from a male flower were teased apart in 100
ll of Alexander’s stain. The stained pollen grains in
aliquots of the pollen suspension were counted on
a Fuchs-Rosenthal hemacytometer, and appropriate
123
118 Plant Cell Tiss Organ Cult (2008) 95:115–124

calculations were done to determine the approximate Statistical analysis

number of stained pollen grains in the whole flower.
‘‘Pollen number’’ in the same samples was deter-
mined by the methods described above except that
the average microscope slide count was multiplied by
2.5 to compensate for the fact that the anthers were
crushed in 100 ll of stain rather than in 40 ll.
Flow cytometry
The statistical analysis for the categorical data was
modified from Alan et al. (2004). A logistic regres-
sion model was used since the dependent variable
was dichotomous. The analyses were performed
using the genmod procedure (SAS Institute 2004).
The mean separation was performed by least square
means.

Ploidy was assessed by flow cytometry (Lotfi et al.

2003). Young leaves on the second node from the top
were collected from in vitro melon plantlets. Leaves of
in vitro grown triploid broccoli (Brassica oleracea var.
italica) were used as an internal standard. Melon and
broccoli leaves were chopped together. The ploidy of
the initial parthenogenetic plants was determined
about 4 months after transfer of seeds to liquid E20A
medium. The ploidy of plants after in vitro colchicine
treatment was checked about 3 months after treatment.
Seed germination tests
Seeds were evenly placed on circles of brown paper
towel moistened with water in a glass Petri dishes
(*40 seeds per dish). The seeds were incubated at
31–33°C and scored for germination after 3–4 days.
Results
Recovery of parthenogenetic plants
A total of 15 fruits was obtained from the three
genotypes after pollination with irradiated pollen
(Table 1). Success in fruit set after the pollination
was 30–40%, i.e., about 3 pollinations were required
to obtain a fruit. The pollinations were performed
during two different seasons: summer and winter.
The frequency of embryos available for excision was
significantly greater in summer (2.8%) than in winter
(1.0%).
Of 63 plantlets recovered, 46 (73%) were haploid
and 17 (27%) were mixoploid (Table 2). None was
diploid. Figure 2a shows a typical haploid histogram,

Table 1 Plant recovery from cultured melon embryos excised from seeds formed after pollination with irradiated pollen
Pollination period (2005) Genotype Fruits obtained Seeds cultured Embryos excised Embryos/seeds (%) Plants obtained

June–July 510 3 1732 55 3.2 34

518 2 852 19 2.2 11
521 3 543 12 2.2 5
Total 8 3127 86 2.8a 50
Nov.–Dec. 510 4 1443 16 1.1 9
518 1 200 2 0.0 1
521 2 785 7 0.9 5
Total 7 2428 25 1.03b 15
Total/mean 15 5555 94 1.8 65
Means followed by different letters are significantly different using Tukey tests (P \ 0.05)

Table 2 Ploidy of melon

Genotype Haploid Mixoploid Mixoploid Total
plants obtained after
(1C + 2C) (1C + 2C + 4C)
pollination with irradiated
pollen (%) 510 34 (79) 4 (9) 5 (12) 43
518 8 (62) 3 (23) 2 (15) 13
521 4 (57) 0 (0) 3 (43) 7
Total 46 (73) 7 (11) 10 (16) 63

123
Plant Cell Tiss Organ Cult (2008) 95:115–124 119

Fig. 2 Flow cytometry histograms of melon leaf tissue with triploid broccoli (Br) as internal control, (a) Haploid (b) 1C + 2C + 4C
mixoploid (c) 1C + 2C mixoploid (d) Diploid from seed-grown melon

and Fig. 2d shows a diploid histogram from a seed-
grown plant. The mixoploid plants showed two
patterns of peaks. Figure 2b shows a 1C + 2C + 4C
mixoploid. The 2C peak probably represents both
undivided 2C nuclei as well as dividing 1C nuclei; the
4C peak probably came from dividing 2C cells. The
mixoploid histogram in Fig. 2c shows a large 2C peak
comparable to the 1C peak and no 4C peak.
Effects of treatment with colchicine in vitro
Shoot tip and node explants from haploid and
mixoploid plants were treated with colchicine to
induce chromosome doubling. The survival rate of
colchicine-treated explants varied from 39–83%
depending on the method of application (Table 3).
The 3 cm shoot tips treated with 500 mg/l colchicine
showed a significantly greater survival rate (83%)
than other treatments. The lowest survival rates (39
and 40%) were from node explants. The 3 cm tip
explants treated with 500 mg/l colchicine also
showed significantly better results for conversion to
diploidy: 22% of explants treated (Table 3) and 26%
of surviving explants. Conversion to diploidy in the
other treatments varied from 0 to 7% of explants
treated. About 33 and 11% of the haploid explants
were converted to mixoploids and diploids, respec-
tively, by treatment of 3 cm tips with 500 mg/l
colchicine (data not shown).
Pollen released from anthers of greenhouse-grown
plants was examined microscopically. Untreated
haploid plants and most mixoploid plants had pollen
numbers ranging from 0–3 (data not shown). Pollen
from 72 plants treated with colchicine in vitro was
examined to assess the effects of the treatments
(Table 4). Most of the plants scored as haploid by

Table 3 Ploidy of plants recovered from tip and node explants after 3 h of in vitro treatment with colchicine
Explant Colchicine (mg/l) Explants treated Explants survived (%) Ploidy after treatment
Haploid (%a) Mixoploid (%a) Diploid (%a)

Tip (1.5 cm) 500 103 61 (59)b 33 (32) 23 (22ab) 5 (5b)

1000 108 68 (63)b 40 (37) 20 (19ab) 8 (7b)
Tip (3 cm) 500 165 137 (83)a 49 (30) 52 (32a) 36 (22a)
Node 500 51 20 (39)c 16 (31) 2 (4b) 2 (4b)
1000 50 20 (40)c 13 (26) 7 (14ab) 0 (0b)
Total 477 306 (64) 151 (32) 104 (22) 51 (11)
Means followed by the same letter are not significantly different using pairwise least square means tests (P \ 0.05)
a
Percent of explants treated

123
120 Plant Cell Tiss Organ Cult (2008) 95:115–124

Table 4 Number and frequency of haploid, mixoploid and diploid plants in three pollen number classes [low (\5), intermediate (5–
20), and high ([20)]
Ploidy after treatmenta No. of plants with each pollen number (% of plants)
Total plants \5 5–20 [20

Haploid 7 5 (71) 0 (0) 2 (29)

Mixoploid 47 23 (49) 8 (17) 16 (34)
Diploid 18 9 (50) 2 (11) 7 (39)
Total 72 37 (51) 10 (14) 25 (35)
The plants were derived from explants treated with colchicine in vitro
a
As determined by flow cytometry

flow cytometry had low pollen numbers, but 2/7
(29%) had numbers greater than 20 (hereafter as
[20). Of the 18 plants scored as diploid by flow
cytometry, 7 (39%) had a pollen number [20. A
comparable percentage (34%) of mixoploid plants
had similarly high pollen numbers. Overall, 47 plants
(65%) had pollen numbers \20; however, the mix-
oploids had more pollen numbers between 5 and 20
than the haploids.
Figure 3 shows stained and unstained pollen from
a mixoploid flower. Almost all haploid plants shed no
visible pollen, whereas all plants visibly shedding
pollen had pollen numbers [20. Male flowers with
pollen numbers [20 contained approximately 800
pollen grains, as determined by correlation of hema-
cytometer counts and microscope slide counts of
stained pollen grains from the same preparation (see
materials and methods; data not shown).
A total of 360 pollinations (average of 4.6/plant)
was done on the 72 plants in Table 4, regardless of
ploidy or pollen number. Fruits were obtained from
18 (25%) of them (Table 5). Most of the fruits set on
plants with a pollen number [20. Genotype 510 gave
the best results, with 68% fruit recovery rate for
pollen numbers [20. Fruits of this genotype were
also obtained with pollen numbers of 5–20 and from
a few plants with lower pollen numbers. Most
genotype 518 plants had pollen numbers \5, and no
fruits set on them. One of three plants of genotype
521 with pollen number [20 produced a fruit.
Seeds from 12 fruits were checked for germination
(Table 6). When the pollen number was [20, seed
germination was [50% and [90% for seven of nine
plants. When the pollen number was \5, the seeds in
the fruit did not germinate. Most of the fruits had 10
seed weights of 181–290 mg. Seed weights of four
fruits were much lower (44–90 mg/10 seeds). Three
of these fruits came from plants with pollen numbers
\20.

Table 5 Fruit set [no. of fruit/no. of plants pollinated (%)]

after pollination of plants derived from colchicine treatment
disregarding ploidy but classified by pollen number
Genotype Pollen number
\5 5–20 [20 Total

510 2/20 (10) 2/2 (100) 13/19 (68) 17/41 (41)

518 0/15 (0) 0/8 (0) 0/2 (0) 0/25 (0)
521 0/2 (0) 0/1 (0) 1/3 (33) 1/6 (17)
Fig. 3 Stained and unstained pollen from a mixoploid melon Total 2/37 (5) 2/11 (18) 14/24 (58) 18/72 (25)
plant (1009). The pollen number is 53

123
Plant Cell Tiss Organ Cult (2008) 95:115–124 121

Table 6 Germination of seeds

Plant Genotype Pollen Seeds in Weight/10 No. of seeds tested Germinated
from fruits on plants derived
number fruit seeds (mg) for germination seeds (%)
from explants treated with
colchicine in vitro 1 510-07 \5 294 51 30 0 (0)
2 510-07 \5 186 44 35 0 (0)
3 510-07 5–20 421 45 35 2 (6)
4a 510-01 [20 530 247 35 32 (91)
5a 510-01 [20 335 232 35 34 (97)
6 510-01 [20 158 223 35 32 (91)
7 510-07 [20 55 90 25 13 (52)
a
All plants were derived from 8 510-07 [20 319 270 35 35 (100)
different parthenogenetic 9 510-07 [20 188 181 34 31 (91)
embryos except for plants 4
and 5, which were clones of the 10 510-07 [20 197 249 35 34 (97)
same embryo. They were 11 510-07 [20 115 260 35 35 (100)
cloned prior to colchicine 12 521-08 [20 29 182 12 7 (58)
treatment

Effects of treatment with colchicine in vivo
A total of 132 plants was treated with colchicine in
vivo (Table 7). Of these, 103 (78%) survived.
Flowers on these plants were checked for pollen
number. Flowers from seed-grown plants in the
greenhouse and haploid plants in vitro were used as
positive and negative controls (Table 7). Pollen
numbers in the in vivo-treated haploids and mixop-
loids showed a bimodal distribution, with 97%
having a pollen number of either \5 or [20. All
flowers from shoots exposed to 1000 mg/l of
colchicine for 12 h had pollen numbers of \5, like
flowers on haploid plants. Pollen numbers for the 2 or
4 h treatments with 5000 mg/l colchicine were greater
and statistically equivalent. In these treatments, 19
and 24% of the plants had a pollen number [20.
Among the seed-grown diploid control plants, 94%
had pollen numbers [20.
In the greenhouse, many of the plants from in vivo
colchicine treatments showed an abnormal growth
pattern, designated ‘‘cluster syndrome.’’ Melon
leaves are normally attached to the stem alternatively
with elongated internodes. The clustered leaves were
densely attached around the stem with short petioles
and few flowers (Fig. 4). Of the ten plants from the
1000 mg/l colchicine treatment and the 63 plants
from the 5000 mg/l treatment, 60% showed the
cluster syndrome. These abnormalities were also
observed in 28% of culture-derived plants never
exposed to colchicine and transferred to the green-
house in the winter. Cluster syndrome was rare in
plants from explants treated with colchicine in vitro
and transplanted during the summer (*5% of 72
plants). It was never seen in standard seed-grown
melon (M. Falise, personal communication).
All of the 103 plants surviving after treatment with
colchicine (Table 7) were self-pollinated (average of 5

Table 7 Effect of in vivo colchicine treatment on plant survival and pollen number
Colchicine (mg/l) Duration (h) Plants treated Plants survived (%) Pollen number (%) Average pollen number
\5 5–20 [20

1000 12 16 12 (75) 12 (100) 0 (0) 0 (0) 1c

5000 2 69 57 (83) 43 (75) 3 (5) 11 (19) 9b
4 47 34 (72) 25 (74) 1 (3) 8 (24) 10 b
Total 132 103 (78) 80 (78) 4 (4) 19 (18)
Controls
Haploid in vitro – 31 31 (100) 0 (0) 0 (0) 0c
Diploid (seed grown) – 17 0 (0) 1 (6) 16 (94) 25 a

123
122
Fig. 4 (a) Top of a melon
plant with cluster syndrome.
(b) Leaf of plant with
cluster syndrome. (c) Leaf
of normal plant
pollinations/plant). Four fruits were recovered. Three
were from the 5000 mg/l 2 h treatment (5.2% of the 57
surviving plants without cluster syndrome) and one
from the 5000 mg/l 4 h treatment (2.9% of the 34
surviving plants). All of these fruits came from plants
with high pollen numbers. The fruits from the in vivo
treatments were smaller (48–300 g) than those from in
vitro treatments with colchicine (300–1000 g).
Discussion
Culture of seeds from fruits obtained by pollination
with irradiated pollen in liquid medium allowed
convenient recovery of parthenogenetic melon plants.
These were easily cloned by nodal cuttings to
increase the population of plants. The majority of
our parthenogenetic plants was haploid, as in earlier
reports (Ficcadenti et al. 1995; Cuny et al. 1993;
Yashiro et al. 2002; Lotfi et al. 2003). We detected no
spontaneous doubling, even after repeated checks of
ploidy by flow cytometry. Chromosome doubling was
therefore required to produce fertile plants.
We did not observe genotypic variation for
chromosome doubling; genotypes 510, 518 and 521
123
Plant Cell Tiss Organ Cult (2008) 95:115–124
showed similar ploidy changes after colchicine
treatments (data not shown). This result agrees with
Lotfi et al. (2003). Yashiro et al. (2002) reported that
five melon cultivars showed different chromosome
duplication rates after being sprayed with 1000 mg/l
colchicine in vivo; however, their sample sizes were
small (2 to 11 plants/cultivar).
Previous reports about in vitro colchicine treat-
ment of parthenogenetic melon plants are limited.
Lotfi et al. (2003) recovered 12% diploids from
treatments similar to ours. Our best duplication rate
was 22% for 3 cm tips treated for 3 h with 500 mg/l
colchicine. Claveria et al. (2005) reported the
survival rate of cucumber treated in vitro with 500
lM (*200 mg/l) colchicine for 48 h was 20–60%,
and the duplication rate was 30%. Koksal et al.
(2002) and Yetisir and Sari (2003) tested several
different in vivo treatments for chromosome dupli-
cation in muskmelon. Dipping shoot tips in 5000 mg/l
colchicine for 2 h produced about 90% diploids;
however, no fruit recovery was reported. Yashiro
et al. (2002) sprayed lateral shoot tips from 5 haploid
genotypes with 1000 mg/l colchicine + detergent and
obtained 0–100% doubled haploids, depending on the
genotype. The best duplication rate in our in vivo
Plant Cell Tiss Organ Cult (2008) 95:115–124
experiments was 19–24%, as measured by pollen
number of resulting plants.
Pollen number was correlated with fruit set and
seed viability. Similarly, Winsor et al. (1987)
reported that Cucurbita pepo plants with low (240
pollen grains/flower) and high pollen load (stigmatic
surface saturated with pollen) varied for seed pro-
duction (39.5 seeds/fruit vs. 375.9 seeds/fruit,
respectively). C. foetidissima needed *4.3 pollen
grains to produce a single mature seed, so that *900
pollen grains were necessary for a full complement of
*200 seeds (Winsor et al. 2000). Sanford (1992)
reported that deposition of 1000 pollen grains on the
stigma of watermelon was required for production of
a uniform melon. In DH work, a full complement of
seeds is not essential. Our pollen number of [20 was
equivalent to *800 fertile pollen grains per flower,
sufficient for good fruit and seed production.
There is little previous information about the
efficiency of fruit set after colchicine treatments and
no previous report about the germination of seeds
from DH melons, a key step in the recovery of DH
lines. Fruits on plants with high pollen numbers
usually had high seed weight and good germination
(Table 6). Although we obtained a few fruits from
plants with pollen number \5, the fruits had small
seeds that did not germinate under standard condi-
tions. When the seed coats of such seeds were
removed, germination was often increased (data not
shown). Seed coat removal may therefore allow
recovery of viable progeny even from poor quality
fruits/seeds. If a few seeds germinate and produce
fertile plants, recovery of the next generation for seed
production requires only about 3 months.
We used two approaches to evaluate the efficacy
of chromosome doubling procedures: flow cytometry
and pollen counts. Flow cytometry provides a direct
measurement of the percentage of nuclei of different
ploidy and can be applied to treated plants at any
stage of growth. However, flow cytometry requires
experience in complex sample preparation, is expen-
sive and often not readily available. A further concern
is that flow cytometry does not distinguish euploids
from aneuploids so many plants with a diploid (2C)
peak may actually have aneuploid chromosome
numbers. Only 39% of the plants scored as diploid
by flow cytometry had pollen numbers [20, and only
28% actually produced fruits, even after repeated
pollinations (data not shown). Counts of pollen
123
number are simple and inexpensive. Plants with
pollen numbers [20 gave fruit recovery rates of 58%
(Table 5). On the other hand, pollen number cannot
be determined until the treated plants have male
flowers, so considerable greenhouse space is required
for several months unless the plants flower in vitro, as
sometimes happens (Lotfi et al. 2003). Thus the best
approach depends on the resources and space
available.
Our comparison of in vivo and in vitro colchicine
treatments was based on pollen numbers. As
expected, haploid plants had little or no stained
pollen. In vitro treatment of 3 cm shoot tips with 500
mg/l colchicine for 3 h was selected for comparison
because flow cytometry indicated the highest dupli-
cation rate. For the in vivo treatment, 2 and 4 h
treatments with 5000 mg/l colchicine were combined
because they showed no significant difference in
pollen numbers. The in vitro treatments were more
effective, giving 38% of plants with pollen number
[20, as opposed to 21% for the in vivo treatments,
respectively. The chi square test shows the p value is
0.023. The higher rate of recovery of plants with
pollen numbers [20 means a better chance of fruit
set. For better duplication rate, a longer duration of
colchicine treatment in vitro might be worthwhile.
Increasing the level of colchicine above 500 mg/l was
not beneficial, at least for 1.5 cm tips (Table 3).
Several areas of further work may be appropri-
ate. Recent studies of chromosome doubling
strategies for onion (Alan et al. 2007) showed that
a different anti-mitotic agent, amiprofos methyl
(APM), gave doubling rates comparable to those
obtained with colchicine at substantially lower
concentrations. Colchicine has higher mammalian
toxicity than APM (Alan et al. 2007), so it would
be worthwhile to test the effects of APM on melon
haploids.
A melon plant in vitro has 5–10 nodes and only
one shoot tip. It would therefore be desirable to have
an efficient doubling procedure for nodal explants.
We found that nodes had poor survival and low
doubling rates after 3 h exposure to 500 or 1000 mg/l
colchicine. Since the doubling rate at 1000 mg/l was
lower than at 500 mg/l, increasing the colchicine
concentration is unlikely to be helpful. A longer
duration of exposure to 500 mg/l or alteration of
culture media may enhance survival and doubling of
nodal explants.
123
124
In conclusion, we found that in vitro treatment of
3 cm shoot tip explants gave better survival, chro-
mosome duplication and fruit set than shorter shoot
tips or nodal explants. In vivo treatment of shoot tips
was less effective. Conversion to diploidy (as scored
by flow cytometry) does not guarantee recovery of
fruits, and pollen counts may be as reliable a
predictor of plant fecundity as flow cytometry. If
greenhouse space is not limiting, the simplest strategy
for recovery of DH fruits and seeds from treated
plants is to transfer all plants to the greenhouse and
self-pollinate the ones with visibly shed pollen. When
only a few parthenogenetic plants are available, it
may be worthwhile to self-pollinate all plants, since
some fruits with viable seeds may set even when
pollen production is minimal.
Acknowledgements We thank Dr. Molly Jahn for providing
the starting melon plant materials and Matt Falise for
assistance with the plants in the greenhouse. Financial
support was provided by the Cornell Vegetable Breeding
Institute and by Hatch Project 149–422.
References
Alan AR, Lim W, Mutschler MA, Earle ED (2007) Comple-
mentary strategies for ploidy manipulations in gynogenic
onion (Allium cepa L.). Plant Sci 173:25–31. doi:10.1016/
j.plantsci.2007.03.010
Alexander MP (1969) Differential staining of aborted and non-
aborted pollen. Stain Technol 44:117–122
Claveria E, Garcia-Mas J, Ramon D-S (2005) Optimization of
cucumber doubled haploid line production using in vitro
rescue of in vivo induced parthenogenic embryos. J Am
Soc Hortic Sci 130:550–560
Cuny F, Grotte M, Dumas de Vaulx R, Rieu A (1993) Effects
of gamma irradiation of pollen on parthenogenetic haploid
production in muskmelon (Cucumis melo L.). Environ
Exp Bot 33:301–312. doi:10.1016/0098-8472(93)90076-R
Doré C, Boulidard L, Sauton A, Rode J-C, Cuny F, Nie-
mirowiecz-Szczytt K et al (1995) Interest of irradiated
pollen for obtaining haploid vegetables. Acta Hortic 392:
123–128
Ficcadenti N, Veronese P, Sestili S, Crino P, Lucretti S,
Schiavi M et al (1995) Influence of genotype on the
123
Plant Cell Tiss Organ Cult (2008) 95:115–124
induction of haploidy in Cucumis melo L. by using irra-
diated pollen. J Genet Breed 49:359–364
Ficcadenti N, Sestili S, Annibali S, Di Marco M, Schiavi M
(1999) In vitro gynogenesis to induce haploid plants in
melon Cucumis melo L. J Genet Breed 53:255–257
Koksal N, Yetisir H, Sari N, Abak K (2002) Comparison of
different in vivo methods for chromosome duplication in
muskmelon (Cucumis melo). Acta Hortic 588:293–298
Lotfi M, Alan AR, Henning MJ, Jahn MM, Earle ED (2003)
Production of haploid and doubled haploid plants of melon
(Cucumis melo L.) for use in breeding for multiple virus
resistance. Plant Cell Rep 21:1121–1128. doi:10.1007/
s00299-003-0636-3
Sanford MT (1992) Beekeeping: watermelon pollination.
Document RF-AA091, Institute of Food and Agricultural
Sciences, University of Florida, USA
Sari N, Abak K (1994) Induction of parthenogenetic haploid
embryos after pollination by irradiated pollen in water-
melon. HortScience 29:1189–1190
SAS Institute (2004) SAS/STAT 9.1. User’s guide. SAS
Institute Inc., Cary, USA
Sauton A (1988) Effect of season and genotype on gynogenetic
haploid production in muskmelon, Cucumis melo L. Sci
Hortic (Amsterdam) 35:71–75. doi:10.1016/0304-4238
(88)90038-6
Sauton A, Dumas de Vaulx R (1987) Induction of gynogenetic
haploid plants in muskmelon (Cucumis melo L.) by use of
irradiated pollen. Agronomie 7:141–148. doi:10.1051/
agro:19870209
Sauton A, Olivier C, Chavagnat A (1989) Use of soft X-ray
technique to detect haploid embryos in immature seeds of
melon. Acta Hortic 253:131–135
Sheldrake R, Boodley JW (1973) Cornell peat-like mixes for
commercial plant growing. Cornell Cooperative Exten-
sion Bull 1.B:43
Winsor JA, Davis LE, Stephenson AG (1987) The relationship
between pollen load and fruit maturation and the effect of
pollen load on offspring vigor in Cucurbita pepo. Am Nat
129:643–656. doi:10.1086/284664
Winsor JA, Peretz S, Stephenson AG (2000) Pollen competi-
tion in a natural population of Cucurbita foetidissima
(Cucurbitaceae). Am J Bot 87:527–532. doi:10.2307/
2656596
Yashiro K, Hosoya K, Kuzuya M, Tomita K, Ezura E (2002)
Efficient production of doubled haploid melon plants by
modified colchicine treatment of parthenogenetic hap-
loids. Acta Hortic 588:335–338
Yetisir H, Sari N (2003) A new method for haploid muskmelon
(Cucumis melo L.) dihaploidization. Sci Hortic (Amster-
dam) 98:277–283. doi:10.1016/S0304-4238(02)00226-1