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Induction of tetraploidity with antimicrotubule agents

in oriental melon (Cucumis melo var. Makuwa)
a a a a
Kang Wang , Linchi He , Honglang Yan & Xiaoyun Wei
a
Jiangsu Riverine Institute of Agricultural Sciences, Xueyao, Rugao, Jiangsu 226541, P.R.
China
Published online: 14 Aug 2015.

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To cite this article: Kang Wang, Linchi He, Honglang Yan & Xiaoyun Wei (2015): Induction of tetraploidity with
antimicrotubule agents in oriental melon (Cucumis melo var. Makuwa), Israel Journal of Plant Sciences, DOI:
10.1080/07929978.2015.1067411

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 Israel Journal of Plant Sciences, 2015
http://dx.doi.org/10.1080/07929978.2015.1067411

Induction of tetraploidity with antimicrotubule agents in oriental melon

(Cucumis melo var. Makuwa)
Kang Wang, Linchi He*, Honglang Yan and Xiaoyun Wei
Jiangsu Riverine Institute of Agricultural Sciences, Xueyao, Rugao, Jiangsu 226541, P.R. China
(Received 8 June 2015; accepted 25 June 2015)

Polyploidy induction is an important method for the innovation of germplasm resources because polyploid plants have
superior characteristics to diploid ones. In this paper, oriental melon (Cucumis melo var. makuwa) tetraploids were induced
Downloaded by [Nanyang Technological University] at 10:53 20 August 2015

by treating the apical meristem of seedlings with oryzalin, amiprophos-methyl (APM) and colchicine. The results showed
that each of these three antimicrotubule agents could induce the formation of tetraploid oriental melon, but oryzalin and
APM showed higher efficiencies of tetraploid induction. The efficiencies of tetraploid induction were highest (14.44%)
when treated with 80 mmol¢L¡1 APM for 6 days. Stomata characteristics were used to screen the treated plants for changes
in ploid levels preliminarily. Subsequently, the changes in ploid levels were further confirmed by flow-cytometric analysis
and chromosome counts of root tip cells. The comparison between diploids and tetraploids in fruit characteristics,
reproductive characteristics, and postharvest storage qualities showed that the fruit of tetraploids had thicker flesh, higher
total soluble solid (TSS) content and lower fruit shape index than diploids. The pollen grains of tetraploids were larger but
showed lower vitality. In addition, a higher percentage of embryo-deficient seeds contributed to lower fertility. During
postharvest storage, the fruit of tetraploids softened faster, had higher TSS content and lower respiration rate.
Keywords: oryzalin; amiprophos-methyl (APM); colchicine; fruit quality; reproductive characteristics; postharvest storage
quality

Introduction morphological, biochemical and physiological characters

Melon (Cucumis melo L.) contains eight cultivated
varieties, namely cantalupensis, chito, conomon, dudaim,
inodorus, flexuosus, reticulatus, and makuwa (Choi et al.
2012). Oriental melon (Cucumis melo var. makuwa) origi-
nated from the Middle East and Asia, and is one of the
most commercially important variants in the Chinese,
Korean and Japanese markets. The fruits of this variety
usually have thin rind, high sugar and juice contents and
little odor. However, the thin mesocarp, small fruit size
and short postharvest shelf-life affect the commercial
value of oriental melon.
Polyploidy induction is an important method for the
innovation of germplasm resources. Generally, polyploid
plants show superior characteristics to the diploid progeni-
tor in morphological changes, genetic adaptability and tol-
erance to environmental stresses (Xiong et al. 2006). In
addition, tetraploid plants of fruit crops are used as breed-
ing parents to produce triploid plants (Jaskani & Khan
2000). Previous studies of tetraploid melons focused on
the muskmelon or honeydew melon (Batra 1952; Fassu-
liotis & Nelson 1992; Zhang et al. 2010; Ren et al. 2013).
However, limited research has been conducted on C. melo
var. makuwa, which differs from other varieties in
(Liu et al. 2004a).
Colchicine has traditionally been used to induce chro-
mosome doubling in many species, such as citrus (Aleza
et al. 2009), hypericum (Meyer et al. 2009), oncidium
(Cui et al. 2010) and rhododendron (Hebert et al. 2010).
However, colchicine is very toxic to humans because it
binds specifically to animal tubulin dimers and inhibits
microtubule polymerization during mitosis and meiosis
(Petersen et al. 2003). Recent reports showed that some
antimitotic agents, such as amiprophos-methyl (APM),
oryzalin and trifluralin, were less toxic and more efficient
in inducing chromosome doubling in plants than colchi-
cine (Petersen et al. 2003; Thao et al. 2003; Zlesak et al.
2005; Dunn and Lindstrom 2007).
In this study, oryzalin, APM and colchicine were
applied to oriental melon to assess the potential of these
three antimicrotubule herbicides for induction of tetra-
ploidy. The ploidy level of treated plants was determined
by stomatal characteristics, flow-cytometric analysis and
chromosome counts in root tip cells. Because oriental
melon is a fruit crop variety, fruit and reproductive
characteristics, and postharvest storage quality of fruit
between tetraploid and diploid plants were also compared

*Corresponding author. Email: 373033410@qq.com

Ó 2015 Jiangsu Riverine Institute of Agricultural Science

 2 K. Wang et al.
to evaluate the potential value of tetraploids for commer-
cial production. Our overall objective was to improve the
commercial characteristics of oriental melon.
Material and methods
Plant material
Oriental melon “OM-1226” is an elite inbred line with
white flesh and a smooth rind surface, which has been
inbred for more than 10 generations at the Jiangsu River-
ine Institute of Agricultural Sciences, Nantong, Jiangsu,
China.
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Induction of tetraploid oriental melon with antimicrotu-
bule and antimitotic agents
Dry seeds of oriental melon were immersed in tap water
for 6 h at room temperature. Then the seeds were placed
on filter paper moistened with distilled water and germi-
nated in darkness at 24  C for 36 48 h. The germinated
seeds were transferred to 50-cell plug trays containing a
mixture of peat and perlite medium (3:1, v/v). The trays
were covered with transparent plastic film and incubated
under greenhouse conditions. Different concentration gra-
dients of colchicine (5000, 10,000 and 15,000 mmol¢L¡1),
oryzalin (20, 40, 80 and 120 mmol¢L¡1) and APM (20, 40,
80 and 120 mmol¢L¡1) were applied to the apical meri-
stem of oriental melon seedlings with expanded cotyle-
dons. The treatments were carried out under greenhouse
conditions with different shading durations (70% shading
rate for 4, 6 and 8 days). Control seedlings were similarly
treated with cotton balls saturated with distilled water.
Each treatment was applied to 30 seedlings and this was
replicated three times. After treatment, the shade facilities
were removed and the plants were thoroughly washed
with water.
Preliminary screening for putative tetraploids by evalua-
tion of leaf stomatal characteristics
Because a large population of seedlings was generated
from the apical tip application method, it was necessary to
isolate a smaller population of putative tetraploids. Stoma-
tal characteristics, consisting of stomata size, stomata den-
sity, and the number of chloroplasts in guard cells, have
proved to be reliable indicators of ploidy in many varieties
(Gu et al. 2005; Stanys et al. 2006; Liu et al. 2007). Those
seedlings with larger stomata, lower stomata density, and
a higher number of chloroplasts in guard cells were
selected as putative tetraploids. Leaf samples were taken
from the third true leaf below the shoot apex. The epider-
mal cell layer from the abaxial surface of leaves was
stripped with tweezers, placed on a microscope slide,
stained with a 1% w/v I2-KI solution, covered with a
cover slip, and observed under a light microscope. The
stomata size, stomata density, and the number of chloro-
plasts in guard cells of 10 randomly selected putative pol-
yploids were measured under a Nikon microscope (E100,
Tokyo, Japan), based on 20 cells from three different
leaves per individual.
Determination of ploidy levels by flow cytometry
The ploidy levels of putative polyploids were identified
with a flow cytometer (BD Biosciences FACS Calibur,
America). Following procedures based on Urwin et al.
(2007) with modifications, tissue samples taken from
young leaves (1 g) were chopped with a razor blade and
immersed in 500 mL Galbraith’s extraction buffer for
1 2 min. After filtration through a 30-mm Cell-Trics
disposable filter to remove cell debris, 150 mL staining
solution containing the dye 3, 8-diamino-5-[3-(diethylme-
thylammonio) propyl]-6-phenylphenanthridinium diio-
dide (PI) was added. The samples were then analyzed
with the flow cytometer. Ten leaves were collected from
diploid plants to determine the standard peak of diploid
cells.
Determination of ploidy levels by chromosome counts
The ploidy level of each plantlet was ultimately confirmed
by chromosome counts of root tip cells from germinated
seeds, which were obtained by self-pollination of each
putative tetraploid and control plants. According to the
method described by Tang et al. (2010) with minor modi-
fications, root tips of 2 3 mm length were excised and
immersed in 2 £ 10¡3 mol¢L¡1 8-hydroxyquinoline for
3 h at room temperature following seed germination.
Subsequently, the root tips were rinsed in water and then
fixed in fresh Carnoy’s fluid (alcohol:acetic acid, 3:1, v/v)
at room temperature for 24 h. The fixed root tips were
stored in 70% ethanol at 4 C until examination. The root
tips were rinsed with water 3 times, then immersed in 1 M
HCl for 10 min at 60 C, and rinsed with water an addi-
tional three times. The root tips were cut transversely,
stained using modified carbolfuchsin (Kao 1975), covered
with a slide and warmed slightly over a flame. Samples
were observed under a Nikon microscope (E100, Tokyo,
Japan) and photographed with a Zeiss camera.
Measurement of fruit characteristics for diploid and
tetraploid plants
Six important fruit characteristics, including fruit weight
(g), flesh thickness (cm), fruit length (cm), fruit diameter
(cm), fruit shape index (length:diameter ratio) and total
soluble solids (TSS) content, were measured for diploid
 Israel Journal of Plant Sciences
and tetraploid plants. For TSS content, juice taken from a
mature fruit was analyzed using a hand refractometer
(Atago, PAL-1, Tokyo, Japan). A total of 10 plants were
tested for each of the diploid and tetraploid plants.
Observation of reproductive characteristics for diploid
and tetraploid plants
Pollen grain size and pollen viability were examined for
diploid and tetraploid plants. For the measurement of pol-
len grain size, tripped flowers were removed and pollen
grains were stained with a 1% w/v I2-KI solution (add:
concentration), then observed and photographed under a
light microscope at a magnification of 200£. The mea-
surement of pollen viability was performed as described
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by Zhong et al. (1999). Pollen grains were cultured in the
medium (2% sucrose, 0.01% boric acid, pH 7.0) for 24 h
at 25 C and the number of germinated pollen grains was
recorded. Pollen grains were considered germinated if the
pollen tube length was longer than the diameter of the
grain. Ten flowers for five diploid and tetraploid plants
were selected for measurement. In addition, seed length,
seed width and the embryo development were measured
from 10 fruits for diploid and tetraploid plants.
Evaluation of postharvest storage quality for diploid and
tetraploid plants
Fruits were detached from vines 30 days after anthesis and
stored at 10 C. Five fruits in triplicate for each parameter
were analyzed for both diploid and tetraploid fruit. Fruit
weight was measured after 0, 3, 6, 9 and 12 days of stor-
age. The percentage weight loss of fruits was calculated
as: weight loss (%) D (Wi ¡ Wf)/Wi £ 100, where Wi is
the sample weight before storage and Wf is the sample
weight after storage. Fruit firmness was measured after 0,
3, 6, 9 and 12 days of storage, as described previously by
Liu et al. (2012). The TSS content was measured after 0,
3, 6, 9 and 12 days of storage, as described above. Respi-
ration rate was measured by a static method as described
by Ferrari et al. (2013) using a CO2 infrared analyzer
(GXH-3051, Beijing, China). Postharvest fruit from dip-
loid and tetraploid plants were placed in a closed box. Gas
sampling was performed 1 h after closing the box. The
respiration rate of the fruit was determined in triplicate
after 0, 2, 4, 6, 8, 10 and 12 days of storage and expressed
in milliliters of CO2 per kilogram per hour.
Statistical analysis
The differences between means were analyzed using
Student’s unpaired t-test (two-sample t-test) or Duncan’s
multiple range test, and 5% probability level was consid-
ered significant. The analysis was performed using SPSS
software (SPSS 19.0.0.329, Inc., Chicago, IL, USA).
3
Results
The efficiencies of tetraploid induction with oryzalin,
APM and colchicine
For all three agents, the percentage survival of plants was
influenced by the agent concentration and treatment dura-
tion. Broadly, the number of surviving plants declined
with increasing concentration at the same treatment dura-
tion, whereas the percentage survival decreased with the
increase in treatment duration for a given agent concentra-
tion (Figure 1a c). With regard to oryzalin, a treatment
concentration of 120 mmol¢L¡1 for 8 days resulted in the
lowest percentage survival (15.56%; Figure 1a). Similar
results were obtained for APM and colchicine treatments
(Figure 1b,c).
Oryzalin applied at 40 mmol¢L¡1 for 6 days generated
the highest tetraploid induction frequency of 11.11%
(Figure 1g). Treatment with APM at 80 mmol¢L¡1 for
6 days yielded the highest induction frequency of 14.44%
(Figure 1h), while treatment with colchicine at 1 £ 104
mmol¢L¡1 for 6 days resulted in 8 tetraploids out of 90
plants examined (Figure 1i). Compared with colchicine
treatment (2.84%), oryzalin and APM showed higher total
frequencies of tetraploid induction (3.43% and 3.52%,
respectively) at a significantly lower concentration
(Figure 1g i). Therefore, oryzalin and APM showed
higher efficiencies of tetraploid induction in oriental
melon.
Ploidy determination
To reduce the number of plants subjected to time-consum-
ing chromosome counts and costly flow-cytometric analy-
sis, preliminary screening was conducted to identify
putative polyploids among the surviving plants treated
with the agents. Significant differences in leaf stomata
characteristics were observed between untreated and
some treated plants, which were designated putative tetra-
ploids. The stomata length, stomata width and chloroplast
number in guard cells of putative tetraploids were signifi-
cantly increased by 62.09%, 50.2% and 99.42%, respec-
tively (Figure 2a,b), whereas the stomata density was
decreased by 56.87% when compared with those of the
control diploids (Table 1).
Flow-cytometric analysis was applied to confirm the
ploidy levels of putative tetraploids after preliminary
screening. Control plants with 2c DNA produced a peak at
the position (channel 500) consistent with analysis of
standards known to be diploid, whereas tetraploids contain-
ing 4c DNA produced a density plot with a peak aligned at
channel 1000 (Figure 3). A total of 98 plants were identi-
fied as tetraploids after flow-cytometric analysis.
Subsequently, the chromosomes in root tip cells were
counted for further confirmation of the ploidy levels. The
chromosome number of the control plants (diploid) was
 4 K. Wang et al.
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Figure 1. Effect of oryzalin, amiprophos-methyl (APM), and colchicine on tetraploid induction of oriental melon. (a c) Effect of ory-
zalin, APM, and colchicine on survival rate, respectively; (d f) effect of oryzalin, APM, and colchicine on putative tetraploids, respec-
tively; (g i) effect of oryzalin, APM, and colchicine on chromosome doubling frequency, respectively. Lowercase letters above the bar
indicates a significant difference at the 0.05 level as determined by Duncan’s multiple range test.

2n D 2x D 24, whereas that of the 98 autotetraploid plants attractions than diploid fruits (Figure 2e,f). Although the
was 2n D 4x D 48 (Figure 4). mean weight of tetraploid fruit was higher than that of
diploids, the difference was not significant.

Characteristics of tetraploid plants Reproductive characteristics

Fruit characteristics
To assess the commercial and breeding potential of orien-
tal melon tetraploids, important fruit characteristics were
compared between the confirmed tetraploids and diploids
in Table 2. Compared with those of diploids, flesh thick-
ness, horizontal fruit diameter and TSS content of tetra-
ploids were significantly increased by 31.3%, 17.9% and
8.1%, respectively, which indicated that the quality of tet-
raploid fruits was enhanced. In contrast, the fruit longitu-
dinal diameter and shape index of tetraploids were
significantly decreased by 10.6% and 23.4%. The lower
shape index of tetraploid fruits resulted in the fruit form
of almost round, conferring tetraploid fruits more
Oriental melon tetraploid produced significantly higher
quantities of pollen (increased by 33.1%) than the corre-
sponding diploid lines (Figure 2c,d). However, the pollen
vitality of tetraploids was weaker, with the percentage
pollen germination significantly decreased by 48.4% com-
pared with that of the diploids (Table 3). In addition, a
high percentage (76.53%) of empty-embryo seeds was
formed in the fruits of tetraploids, indicating poor fertility.
The seed width and length to width ratio of tetraploids
were significantly increased by 11.3% and decreased by
13.2%, respectively, compared with those of the diploids.
Consistent with the fruit, seeds of tetraploid plants were
also flatter than those of the diploids (Figure 2g).
 Israel Journal of Plant Sciences 5
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Figure 2. Stomata, pollen grains, fruit, and seeds of diploid and tetraploid plants of oriental melon. Stomata of (a) diploid plant and (b)
tetraploid plant. Pollen grain of (c) diploid plant and (d) tetraploid plant. (e) Fruit of diploid (left) and tetraploid (right) plants. (f) Meso-
carp of diploid (left) and tetraploid (right) plants. (g) Seeds of diploid (upper) and tetraploid (lower) plants. For a d, scale bar D 10 mm.
For e g, scale bar D 1 cm.

Table 1. Stomata characteristics of putative tetraploid and diploid oriental melon plants.

Stomata characteristic Diploid Putative tetraploid

Stomata length (mm) 27.25 § 0.83 44.17 § 1.56

Stomata width (mm) 19.98 § 0.33 30.01 § 1.03
Stomata density (no./observation field)a 15.65 § 0.68 6.75 § 0.59
No. of chloroplasts in guard cells 8.70 § 0.44 17.35 § 0.63

Each value represents mean § SE.


Indicates a significant difference between the diploid and the putative tetraploid at the 0.01 level as determined by t-test.
a
The density of stomata was measured with an optical microscope at a magnification of £400.

Figure 3. Flow-cytometric analysis of nuclei from (a) diploid and (b) tetraploid plants.
 6 K. Wang et al.

Figure 4. Chromosomes in root tip cells of diploid (a, 2n D 2x D 24) and tetraploid (b, 2n D 4x D 48) plants. Scale bar D 10 mm.
Downloaded by [Nanyang Technological University] at 10:53 20 August 2015

Table 2. Fruit characteristics of diploid and tetraploid oriental Fruit firmness decreased during storage in both ploidy
melon. groups (Figure 5a). The firmness of tetraploid fruit was
significantly higher after storage for 6 days and decreased
Fruit characteristic Diploid Tetraploid more gradually than that of diploid fruits, which declined
Fruit weight (g) 601.4 § 18.60 642.4 § 57.26 sharply from days 6 to 9 of storage. Ultimately, after stor-
Flesh thickness (cm) 2.62 § 0.10 3.44 § 0.13 age for 12 days, the firmness of diploid and tetraploid fruit
Fruit horizontal diameter (cm) 8.64 § 0.21 10.19 § 0.49 decreased by 52.04% and 39.08%, respectively.
Fruit longitudinal diameter (cm) 9.58 § 0.38 8.65 § 0.39 The percentage weight loss of diploid fruits was
Fruit shape index 1.11 § 0.05 0.85 § 0.02 higher than that of tetraploid fruits throughout the 12-day
Total soluble solid content (%) 12.65 § 0.26 13.68 § 0.20 storage period (Figure 5b). However, no significant differ-
ences were observed between the two ploidy groups
Each value represents mean § SE. except at day 9, when the percentage weight loss of dip-
 
, Indicate a significant difference between the diploid and tetraploid at
the 0.05 and 0.01 levels, respectively, as determined by t-test. loid fruit was 2.56% higher than that of tetraploid fruit.
The TSS content rose slightly at first, peaked after
6 days of storage, and then decreased (Figure 5c). The
Postharvest storage quality TSS content of tetraploid fruits had increased by 4.24%
A shortcoming of oriental sweet melon is the short post- on day 6 compared with the initial content, and was higher
harvest shelf life of the fruit because of its typical climac- (but not significantly) than that of diploid fruits (2.43%).
teric behavior and thin pericarp (Liu et al. 2004b). The ultimate decrease in TSS content in tetraploid fruit
Therefore, the postharvest storage quality of tetraploid after storage for 12 days was 2.78%, which was signifi-
fruit was tested to evaluate its storage potential. The dif- cantly lower than that of diploid fruit (10.31%). In addi-
ferences in fruit firmness, percentage fruit weight loss, tion, throughout the storage period, the TSS content of
TSS content and respiration rate showed the same trends tetraploid fruits was significantly higher than that of
between the diploid and tetraploid plants. diploid fruits.
For both diploid and tetraploid fruits, the respiration
rate decreased after 2 days of storage and peaked on day
6, then declined to the lowest value on day 10 (Figure 5d).
Table 3. Reproductive characteristics of diploid and tetraploid Throughout the storage period, the respiration rate of tet-
oriental melon plants. raploid fruit was significantly lower than that of diploid
fruit. The peak respiration rate of tetraploid fruit was
Reproductive characteristic Diploid Tetraploid
70.2% lower than that of diploid fruit.
Pollen grain diameter (mm) 50.81 § 1.47 67.61 § 3.14
Pollen germination (%) 59.64 § 3.36 30.75 § 1.89
Embryo-deficient seeds (%) 22.46 § 3.24 76.53 § 2.22 Discussion
Seed length (mm) 6.98 § 0.04 6.76 § 0.12 The antimicrotubule agents colchicine, oryzalin and APM
Seed width (mm) 3.54 § 0.05 3.94 § 0.03 all induced chromosome doubling of oriental melon in
Seed length:width ratio 1.97 § 0.03 1.71 § 0.03 our study (Figure 1d i). Similar results were reported for
potato (Barandalla et al. 2006), Miscanthus sinensis
Each value represents mean § SE.

Indicates a significant difference between the diploid and tetraploid at (Petersen et al. 2003), and maize (Wan et al. 1991). For
the 0.01 level as determined by t-test. all these agents, the tetraploid induction frequency was
 Israel Journal of Plant Sciences
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Figure 5. Changes in (a) fruit firmness, (b) percentage fruit weight loss, (c) total soluble solids content, and (d) respiration rate during
storage of fruit from tetraploid and diploid oriental melon. The asterisk above a given day denotes a significant difference at p D 0.05
between diploids and tetraploids as determined by t-test.
associated with the durations and concentrations of treat-
ments. For example, the optimal concentration and dura-
tion of oryzalin treatment was 40 mmol¢L¡1 and 6 days,
respectively (Figure 1g). Chromosome doubling was not
induced when the treatment concentration was too low or
the duration was too short. In contrast, the growth of
plants was strongly inhibited if the concentrations of
agents were too high or the durations of treatments were
too long (Figure 1g i). Although many plants were iden-
tified as putative tetraploids, no fruit and seeds were
obtained because of the abnormal growth. In addition, a
number of mutants with a high ploidy level were induced
because of repeated chromosome doubling with a long
treatment duration (data not shown). Thus, the results
indicated that the optimal combination of treatment con-
centration and duration is critical, which was in consistent
with the results of previous studies. Ye et al. (2010)
reported that a concentration of 0.2% colchicine applied
for 96 h or 0.5% colchicine applied for 48 h showed simi-
lar levels of effectiveness for polyploid induction in crape
myrtle. In Calanthe, the most efficient treatment for induc-
tion of tetraploids was 0.003% oryzalin for 1 2 days
(Chung et al. 2014).
In particular, oryzalin and APM induced a higher fre-
quency of tetraploids at a relatively lower concentrations
compared with colchicine (Figure 1g i), which was also
reported previously for potato (Ramulu et al. 1991). The
7
mechanism of increasing ploidy levels is similar for these
agents in that the formation of spindle fibers is blocked
and the normal polar segregation of sister chromatids is
disturbed. However, oryzalin and APM shows higher
affinity with plant tubulins than colchicine does (More-
john et al. 1987), which may lead to a higher efficiency of
chromosome doubling. In addition, there are still some
side effects, including chromosome losses, rearrange-
ments and gene mutations in the polyploidization process
(Luckett 1989). Thus, oryzalin and APM could be consid-
ered the preferable alternative chromosome-doubling
agents to replace of the toxic colchicine for the polyploid
induction of oriental melon.
Several methods can be used to identify the ploidy
levels of mutants. Counting of mitotic chromosomes in
root tip cells is the most direct and accurate tool for ploidy
determination (Dart et al. 2004), but is extremely time-
consuming. Flow-cytometric analysis is a quick but
expensive method to detect the ploidy level. Because a
large population of treated plants were with a diploid
background, it is necessary to develop a screening method
to eliminate diploids and increase the efficiency of tetra-
ploid identification. In previous studies, stomata charac-
teristics were shown to be reliable indicators to
distinguish mutants from diploids (Speckmann et al.
1965; Stanys et al. 2006; Urwin et al. 2007). In the current
study, we reduced the population from 2970 treated plants
 8 K. Wang et al.
to 326 putative tetraploids, which were identified on the
basis of stomata size, stomata density and chloroplast
number in guard cells. Subsequently, 98 tetraploid plants
were confirmed by flow-cytometric analysis and chromo-
some counts. Thus, stomata characteristics analysis was
proved to be a rapid, efficient and economical means of
identifying putative tetraploid plants in oriental melon.
Polyploids are often morphologically larger than the
corresponding diploids and are known in many plant spe-
cies (Gao et al. 2002; Jaskani et al. 2005; Ye et al. 2010).
In the present study, tetraploid oriental melon produced
fruit with a thicker mesocarp and a higher TSS content
(Table 2), as well as higher quantities of pollen grains
(Table 3). The mechanisms leading to novel variation in
newly formed polyploids may include gene dosage effects
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(resulting from the higher number of genome copies), the
uniting of divergent gene regulatory hierarchies, chromo-
somal rearrangements, and epigenetic remodeling, all of
which affect gene content and/or expression levels (Steb-
bins 1950). The lower fruit shape index of tetraploid
plants observed in the present study meant that tetraploid
fruits were flatter than those of diploids (Figure 2e), which
is consistent with the results of Nu
nez-Palenius et al.
(2008) for tetraploid melon. The quality of tetraploid
fruits was improved because of the higher TSS content,
which was in agreement with the report by Zhang et al.
(2010) for tetraploid muskmelon. However, Ren et al.
(2013) observed that fruit shape, size and TSS content of
polyploid plants were similar to those of diploid plants in
honeydew melon. Therefore, different fruit characteristics
may respond differently to tetraploid induction in differ-
ent melon subspecies. Reduced fertility was also observed
in tetraploid oriental melon, which may result from the
low pollen vitality, as described by Ye et al. (2010) for
crape myrtle tetraploids. Abnormal chromosome behavior
in meiosis might be one cause of this phenomenon (Costa
& Forni-Martins 2004).
Fruit firmness, percentage fruit weight loss, TSS con-
tent and respiration rate are important parameters used to
evaluate fruit quality during storage (Bi et al. 2003;
Jin et al. 2013; Ferrari et al. 2013). In the current study,
the firmness of tetraploid fruits declined gradually
(Figure 5a), which indicated that the mesocarp of tetra-
ploid fruits softened more slowly than in diploid fruits.
The fruit weight decreased during storage, which was
associated with respiration and transpiration. However, no
significant differences in fruit weight were observed
between diploid and tetraploid fruits except on day 6
(Figure 5b). With regard to the TSS content, during fruit
ripening the starch in the fruit is hydrolyzed into reducing
sugar, which would lead to the increase in TSS content at
the beginning of the storage period. Then, as a conse-
quence of respiration, the reducing sugar was consumed,
leading to the decrease in TSS content (Wang et al. 1978).
Throughout the storage period in the present study, the
TSS content of tetraploid fruits was significantly higher
than that of diploid fruits (Figure 5c). At the beginning of
the storage period, tetraploid and diploid fruits showed
higher CO2 production, which could be related to the
higher temperature. The lower temperature during storage
contributed to the decreased CO2 production. The respira-
tion rate of tetraploid fruits was significantly lower than
that of diploid fruits throughout the storage period
(Figure 5d). Similar trends have been observed in
Dendrobium (Ketsa et al. 2001) and pear (Sun 1981). The
cell volume of tetraploids is increased because of the mul-
tiple chromosomes, which results in reduction in the cell
surface area to volume ratio, leading to decreased respira-
tion (Pei 1963).
In conclusion, this study demonstrates the effective-
ness of oryzalin and APM as polyploidizing agents in the
successful induction of tetraploids in oriental melon.
Compared with colchicine, oryzalin and APM induced a
higher frequency of tetraploids at a lower concentration
and thus can be considered suitable alternative chromo-
some-doubling agents for the toxic colchicine. Measure-
ment of stomata size, stomata density and the number of
chloroplasts in guard cells can be used as parameters for
preliminary screening of plants with a diploid back-
ground. Subsequently, the ploidy levels can be determined
by flow-cytometric analysis and chromosome counts of
root tip cells. Compared with diploids, tetraploid oriental
melon fruits have a thicker mesocarp, higher center solu-
ble solid content and lower fruit shape index, which also
indicated that fruit quality and type of tetraploids were
improved. However, the lower pollen vitality and higher
percentage of embryo-deficient seeds in the fruit, which
contributed to lower overall fertility, is a serious defect of
tetraploid oriental melon. In addition, tetraploid fruit
showed more gradual softening, lower decreased rate of
TSS content and lower respiration rate during storage. So
the postharvest storage quality of tetraploid fruits was
superior to that of diploid fruits.
Funding
This work was supported by the Independent Innovation Funds for
Agricultural Technology of Jiangsu Province, China (CX(12)5082).
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