Scientia Horticulturae 169 (2014) 14–19

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

An improved method to obtain novel mutants in Cucurbita pepo by

pollen viability
N. Vicente-Dólera a , V. Pinillos b , M. Moya a , M. Del Río-Celestino a , T. Pomares-Viciana a ,
B. Román c, P. Gómez a, *
a
IFAPA Centro La Mojonera. Camino de San Nicolás, 1, 04745 La Mojonera, Almería, Spain
b
Universidad de Almería. Dpt. de Producción Vegetal. Carretera de Sacramento s/n. 04120 La Cañada, Almería. Spain
c
IFAPA Centro Alameda del Obispo. Avd. Menéndez Pidal s/n, 14004, Córdoba. Spain

A R T I C L E I N F O A B S T R A C T

Article history: A new genetic resource for Cucurbita pepo has been developed with chemically induced mutagenesis. The
Received 6 September 2013 seeds of the zucchini cultivar MU-CU16 were treated with 40 mM–80 mM ethyl methanesulfonate (EMS),
Received in revised form 24 January 2014 reaching high germination rates between 70 and 85%. However, most plants of those M1 populations did
Accepted 26 January 2014
not produce offspring, and the fertility rates were lower in plants treated with higher concentrations of
EMS. Once we established that visual flower abnormality rates were not sufficient to explain low fruit
Keywords: yield, pollen viability was analysed with fluorochromatic reaction. Compared with untreated plants,
EMS
treatment with EMS produced a substantial decrease in pollen viability, and only the group of plants with
Mutagenesis
FCR test
pollen viability rates higher than 45% yielded nearly 70% of fruits with seeds. Therefore, the main issues to
Zucchini be addressed for developing mutant lines in this species are to increase the number of mutations in the
Fertility genome and to increase the number of mutant lines with sufficient fertility. In this case, the early plantlet
selection for high pollen viability carried out as part of this work represents a useful tool for use in future
breeding programs by mutagenesis, allowing an increase of up to 40% in the production of mutant lines for
a dosage of 65 mM EMS.
ã 2014 Elsevier B.V. All rights reserved.

1. Introduction particular, provides a random-point mutation wherein G–C base-

Plant populations derived from mutagen-treated germplasm
are potential sources of novel genetic variation. Screening and
selecting for mutants within these populations have added some
valuable new traits to crop gene pools, including short stature and
high-yield rice in China, semi-dwarf durum wheat in Italy and
tomatoes adapted to drought conditions in Cuba (Ahloowalia et al.,
2004).
There are diverse mutagenesis strategies to generate new
genetic variation, but chemical mutagens have gained popularity
because they are easy to use, do not require any specialised
equipment and can provide a very high mutation frequency.
Amongst chemical mutagens, ethyl methanesulfonate (EMS) is
currently the most widely used. Compared with radiation, which
causes damage on a large scale and severely reduces viability
(Sikora et al., 2011), chemical mutagens tend to cause single base-
pair (bp) changes rather than deletions and translocations. EMS, in
* Corresponding author. Tel.: +34 671532004.
E-mail address: pedro.gomez.j@juntadeandalucia.es (P. Gómez).
pairs (bps) are switched to A–T pairs (Hoffmann, 1980). This
chemical-induced strategy has been used to generate new alleles
for nodule development in Lotus japonicus, spike morphology in
barley, lignin and b-glucan production in Avena sativa and extended
shelf life in Cucumis melo (Kurowska et al., 2011).
However, serious difficulties have emerged in the attempt to
produce mutant populations for some crop species, such as
Cucurbita pepo L. This species is perhaps the most polymorphic
with respect to fruit characteristics of the Cucurbitaceae (Duchesne,
1786; Naudin, 1856), comprising eight edible-fruited morphotypes
(Paris, 1986). The zucchini morphotype is the most recently
developed and has the least variability (Paris et al., 2003), but
importantly, it is the most widely consumed by humans. The
breeder’s initial selection of traits for this morphotype included
bush habit, increasing femaleness and early flowering that
increased production and resulted in the first commercial hybrids.
A low number of resistance genes was introgressed in commercial
cultivars from genotypes of related species, such as C. moschata
Duchesne, and wild relatives, such as C. okeechobeensis (small)
Bailey. These species represent an existing source of genetic
variation to incorporate required traits, but their use is limited by

http://dx.doi.org/10.1016/j.scienta.2014.01.045
0304-4238/ã 2014 Elsevier B.V. All rights reserved.
 N. Vicente-Dólera et al. / Scientia Horticulturae 169 (2014) 14–19
crossability barriers, including reduced fertility and quasi-linkage
(Formisano et al., 2010).
Advantageous breeding results may be achieved by efficient
selection methods on mutant populations, which require that the
population size (number of families) compensates for the density of
mutations (Parry et al., 2009). As zucchini plants are relatively large,
the number of plants that can be grown per unit area is limited.
Consequently, there is a need for an efficient screening procedure of
small, young plants. Moreover, self-pollination is necessary to allow
expression of recessive traits, but in allogamous crops having large
plants, such as zucchini, the process of self-pollination and
selection requires considerable field space.
The development of mutant populations is limited by the
reduction in plant fertility often caused by mutagens. Reduced
fertility can be expressed as less fruit and production of fewer seeds
per fruit, forcing an increase in the number of lines to be screened or
the use of milder applications, such as lower concentrations of a
chemical mutagen, to increase fertility, but at the cost of the need
for growing much larger plant populations (Caldwell et al., 2004).
Determining the main parameters that influence fertility in C. pepo,
such as pollen viability analysis, might enable more efficient
production of an adequately sized mutant population. Assessments
of pollen viability, together with artificial pollinations, have proven
useful in monitoring sterility problems (Dent-Acosta et al., 1995;
Rodríguez-Riano and Dafni, 2000) and could, potentially, help
overcome them.
The goal of the present research was to develop an optimal
protocol to establish an EMS mutant population of Cucurbita pepo as
a first step towards efficiently obtaining novel variation in this
species. Selection for increased fruit and seed production based on
selection for increased pollen viability is to be tested as the
foundation of an effective method to generate novel and potentially
useful mutations in zucchini.
2. Materials and methods
2.1. EMS mutagenesis
Experiments were performed using seeds from the Cucurbita
pepo subsp. pepo L. Zucchini Group ‘MU-CU16’. This accession
belongs to the Cucurbita core collection of the Cucurbits Breeding
Group from the Institute for the Conservation and Breeding of
Agricultural Biodiversity (COMAV).
Seeds of ‘MU-CU16’ were treated with EMS. First, to balance the
maximum mutation density with an acceptable plant survival rate,
an EMS “kill curve” analysis was established on batches of 100 seeds
treated with seven different EMS concentrations ranging from
0 mM to 240 mM. The seeds were imbibed in bottles containing
EMS at different concentrations and placed on a rotary shaker
overnight (16 h) at room temperature. To end the EMS treatment,
the seeds were treated with 0.2 mM sodium thiosulfate for 10 min
at room temperature and rinsed with dH2O for 30 min with gentle
shaking. Finally, the seeds were placed on trays over wet paper
overnight prior to planting.
2.2. Mutant population development
Concentrations of EMS deemed suitable for application to C.
pepo seeds were those for which the “kill curve” showed
germination rates higher than 60%. Finally, three optional concen-
trations were applied to produce the mutant populations and
evaluate the effects on offspring production. Because increasing the
EMS concentration decreases the germination rate, the number of
seeds to be treated was increased for the highest EMS dosage
applied. Mutagen-treated plantlets were sown, the plants grew and
controlled self-pollinations were attempted on each plant. Female
15
flowers were protected before anthesis to prevent the transfer of
pollen by insects, and each M1 plant was self-pollinated by hand
early in the morning. At 60–80 days after self-pollination, M2 seeds
were extracted.
The presence of visible mutations in the first mutant generation
was evaluated to determine how mutations altered reproductive
development. To analyse how fertility was prevented in the mutant
population, the plants were classified into three groups: those
producing seeds, those producing fruits without seeds and those
that failed to produce fruits.
2.3. Pollen viability analysis
Male flowers in anthesis from each group of plants were
collected early in the morning and kept in a humid chamber for no
more than 2 h. Pollen viability was determined by the fluorochro-
matic reaction test (FCR test, Heslop-Harrison and Heslop-
Harrison, 1970). Subsequently, pollen grains were immersed in a
freshly prepared solution of fluorescein diacetate made up of a
concentration of approximately 10 6 M in sucrose 0.5 M (Cuevas
and Pinillos, 2008). A few minutes later, pollen grains were
observed under a fluorescence microscope (Olympus BX40F4).
Viability was expressed as a percentage of fluorescent pollen grains,
with at least 200 pollen grains counted per sample.
Pollen viability analysis was carried out in 40 mM and 80 mM
EMS mutant populations because they were the most distant
concentrations of mutagen in the range selected. The fluorochro-
matic reaction (FCR) test was performed on 200 M1 plants
randomly chosen from each population and compared with results
from untreated control plants. Plants were grouped into three
categories depending on pollen viability rates: low, when pollen
viability was less than 45%; medium, when pollen viability was
between 45 and 90%; and high, when pollen viability was higher
than 90%. Seed production was determined by counting seeds
whose shape and size were similar to seeds produced by the
untreated ‘MU-CU16’, which had a germination rate of 100%.
2.4. Selection by pollen viability
An early selection of plants by pollen viability was performed in
two groups of 200 M1 plants treated with 40 mM and 65 mM EMS.
These concentrations were chosen to improve the fruit and seed
recovery from mutant plants in the range of previously selected
concentrations. The groups were then arranged into two random-
ised blocks. Each group was divided into two halves including
plants selected or unselected by pollen viability rates.
Young selected M1 plants were self-pollinated and those having
pollen viability, of the first male flower, of greater than 45% were
compared with unselected plants. Fruits were allowed to mature
and the seeds of each fruit were counted.
The results were subjected to analysis of variance in a 2-factorial
design, one factor being with and without pollen selection and the
other the dosage of EMS. Means of significantly different results
(p < 0.05) were compared with Tukey’s Test.
3. Results
3.1. Production of EMS Cucurbita pepo mutant population
Three concentrations of EMS, 80 mM, 65 mM and 40 mM, were
chosen for large-scale mutagenesis, as they showed germination
rates of approximately 70 to 85% (Fig. 1). Approximately 7000 seeds
that were mutagenized with 80 mM EMS were sown. The resulting
M1 plants were self pollinated but only 6% of the plants produced
seeds (Table 1). Of the approximately 4000 seeds subjected to the
65 mM EMS treatment that were sown, only 9% of the M1 plants
16 N. Vicente-Dólera et al. / Scientia Horticulturae 169 (2014) 14–19
Fig. 1. Titration curve of EMS concentration applied to C. pepo. X-axis: EMS concentrations (mM) applied to seeds. Y-axis: percentage of germinated seeds.
produced seeds. Results with the 4000 seeds subjected to 40 mM
EMS were better, with 34% of the M1 plants producing seeds. The
M2 seeds were collected separately from each M1 plant. Seeds of
1464 M2 families were obtained, 259 from the 80 mM treatment,
231 from the 65 mM treatment, and 974 from the 40 mM EMS
treatment.
3.2. Assessing the mutant population
Most mutations in this population affected vegetative growth.
Some mutations resulted from modifications in the normal
architecture of the plant (Fig. 2a), with changes in leaves
(Fig. 2b–d) that prevented correct flower formation.
Mutations that altered the reproductive development repre-
sented a relatively low quantity. Most common were mutations in
the morphology of floral organs in both floral types. Female flowers
(Fig. 2e) showed changes in one or more whorls at the same time as
well as altered petals (Fig. 2f), stigma (Fig. 2g) and ovary (Fig. 2h).
Male flowers (Fig. 2i) were altered in a similar way (Fig. 2j–l).
However, the observed mutations in reproductive development did
not explain the low fertility rates in this population. Most of the
mutant plants that showed apparently normal male and female
flowers did not form fruits after repeated pollination attempts.
To confirm the success of the mutagenesis, we also investigated
the rate of mutants in the offspring of the first mutant generation at
the seedling stage. Compared with the control group (Fig. 2m),
albino and chlorotic plants (Fig. 2n,o) occurred at a rate of 1% in M2
families derived from treatment with 40 mM EMS. In addition,
changes in plant architecture, dwarfism (Fig. 2p) and delayed
germination were observed in M2 families at low rates.
3.3. Analysis of mutant plant pollen viability
The FCR test showed greater than 90% pollen viability when
applied to 20 untreated plants but lower values in the 40 mM and
80 mM EMS mutant populations, with a great deal of variation in
pollen viability. Only a small subset of the mutant plants reached
the pollen viability values of control plants, 6% in the 40 mM group
and 13.5% in the 80 mM group. The two treatment groups were
Table 1
Summary of the Cucurbita pepo mutant populations developed from several treatments of variable EMS concentrations.
EMS concentration No. mutagenized seeds sown
80 mM 7,000
65 mM 4,000
40 mM 4,000
Total 15,000
comparable in terms of numbers of plants with medium (between
45 and 90%) or low (less than 45%) pollen viability (Table 2).
Compared with control plants, all of which produced fruits with
seeds, production of fruits and seeds was markedly reduced in EMS-
treated plants (Table 2). Over half of the plants having low pollen
viability did not produce fruits and, in the 80 mM treatment, over
half of the remainder had seedless fruits. However, over half of the
treated plants that had medium and high pollen viability produced
fruits and of these a clear majority had seeds.
3.4. Fruit and seed production
Both the dosage of EMS and the plant selection by pollen
viability significantly affected the number of plants that produced
fruits with seeds, and there was a significant interaction between
these two factors (F = 18, DF = 1, P = 0.02). Among the unselected
plants, the higher dosage resulted in a lower incidence of producers
of fruits with seeds (Fig. 3). Among the plants having high pollen
viability, though, dosage had no effect, and at both dosages the
incidence of producers of fruits with seeds was high.
Of the plants treated with 40 mM EMS, selected plants had
nearly double the number of seed-producing fruits, and of the
plants treated with 65 mM EMS, the number was more than
quadruple with an increase in seed-bearing M1 lines of 21 and 40%,
respectively (Fig. 3). The rates of plants without fruit and without
seeds were similar in the selected plants of both groups,
independent of the intensity of the mutagenesis. The effect of
the plant selection also significantly diminished the proportion of
plant without fruits compared with unselected mutant plants.
4. Discussion and conclusion
The first key factor during the establishment of a chemical-
induced mutant population is the identification of the optimal dose
of mutagen. The gentle descending slope on the “kill curve” of M1
germinated seeds was achieved by the broad spectrum of mutagen
concentrations applied to the seed population. According to the
germination curve, assuming cell toxicity in decreasing M1
germination to an accepted level of 70%, and focusing on
No. germinated No. viable seedlings for transplanting No. M2 seed lots
4,900 4,500 259
3,160 2,700 231
3,400 2,900 974
11,460 10,100 1,464
 N. Vicente-Dólera et al. / Scientia Horticulturae 169 (2014) 14–19 17

Fig. 2. Cucurbita pepo phenotypes in mutant populations. a–d. M1 Architectural phenotypes: (a) wild-type, (b) long leafstalk, (c) chlorotic and serrate leaves and (d) bushy
growth habit. e–h. Phenotypes of M1 female flower: (e) wild-type female flower, (f) mutant lacking petals and with unmatured stigma, (g) female flower without stigma and (h)
female flower without stigma and petals. i–l. Phenotypes of M1 male flower: (i) wild-type male flower, (j) open male flower without pollen, (k) male flower with homeotic
conversions of stamens to petals, (l) male flower without a staminal cone. m–p. M2 plants phenotypes at cotyledon stage: (m) wild-type, (n) albino plant, (o) chlorotic leaves
and (p) dwarfism.

maximising mutation rates on the genome (Comai and Henikoff,
2006), the highest EMS dosage permitted for this species was
80 mM. The most suitable treatments were 40 mM and 65 mM, in
which germination rates approximated 80%. Nevertheless, what-
ever dosage was applied, undesirable phenotypes appeared as a
result of the random distribution of mutations in the genome. M2
families were generated from fertile M1 plants segregated for
visible morphological mutations such as dwarfism or changes in
plant architecture (Fig. 2), which also demonstrated the potency of
the mutagen. Albino and chlorotic plants, which correspond to the

Table 2
Percentage of plants that produced fruits in mutant, M1-generation populations of Cucurbita pepo. Values are grouped by low (<45%), medium (45–90%) and high (>90%) pollen
viability for both treatments: 40 mM EMS and 80 mM EMS.

Treatment Pollen viability Plants (%)a Fruit with seeds (%) Seedless fruits (%) Without fruits (%)

Low 44 27.26 9.1 63.64

EMS, 40 mM Medium 50 68 8 24
High 6 66.67 33.33 0
Low 48.5 17.52 29.9 52.58
EMS, 80 mM Medium 38 57.9 36.84 5.26
High 13.5 59.26 40.74 0
Control High 100 100 – –
a
Percentage of total plants in each pollen viability group for both populations.
18 N. Vicente-Dólera et al. / Scientia Horticulturae 169 (2014) 14–19
Fig. 3. Effect of selection by pollen viability on fruit production of mutant plants. Grey columns show the percentages of plants that were previously selected, and dark columns
are percentages of unselected plants. Values are grouped into plants with seed-bearing fruit, plants with seedless fruit and plants without fruit for both treatments: EMS 40 mM
and EMS 65 mM. Histograms with the same letter in the same group did not differ significantly (P  0.05). Error bars represent the standard deviation of the mean.
most frequently observed phenotypes in other well-characterised
mutant collections (Dalmais et al., 2008), also occurred at similar
rates in this study.
The decrease in fertility is the major restriction to obtaining
successive offspring in this species. After treatment with 80 mM
EMS, production of M2 lines was reduced to 6%, but the use of lower
EMS concentrations of 65 mM and 40 mM increased seed produc-
tion only up to 8.5 and 33.5%, respectively (Table 1). A previous
study published on the straightneck morphotype of this species
(Whitwood and Weigle, 1978) used an EMS dose of 35 mM and led
to only one mutant, a dwarf, that had slow development and failed
to produce fruit. Moreover, previous studies on the mutation of
other cucurbit species, such as cucumber or melon (Dahmani-
Mardas et al., 2010; González et al., 2011), showed a decrease in
fertility even with high rates of seed germination. Fertility barriers
seem to be the main problem in establishing mutant populations of
cucurbit species, which share a similar mechanism in determining
male and female flowers throughout development (Peñaranda
et al., 2007). In C. pepo, visible morphological mutations of M1
usually prevent fertility, including mutations that alter male or
female flowers. However, the incidence of those mutations is not
sufficient to explain the problems of fertility in the whole
population.
With lower mutagen dosages, plant mutant populations are
easier to maintain, as reported in mutant barley, for which lethality
and sterility has been avoided using low EMS doses (<40 mM)
(Caldwell et al., 2004). However, reaching a high number of new
alleles in populations with lower mutation rates requires a higher
population size that makes the mutagenesis inefficient. Male and
female fitness must be considered in order to achieve C. pepo
mutant populations that are reproductively successful, and the
estimation of total reproductive fitness based only on the
determination of pollen viability might not be sufficient. Future
evaluations of elements such as pistillate flower number, ovule
number or seed set, will improve the assessment of essential
aspects of the fertilization process. However, pollen viability is a
trait strongly related to fruit set and seed production, with the
additional advantage that determinations can be done at young
stages of the plant. Grouping mutagenized plants of Cucurbita pepo
by pollen viability and comparing seed production demonstrated
the importance of these analyses for offspring production: plants
with low pollen viability (less than 45%) had a reduced incidence of
fruits with seeds. Indeed, for indicators used to define the
parameters of chemical-induced mutation, plant fertility has been
considered more appropriate than the widely used germination
rate (Koornneef 2002; Menda et al., 2004), as has been described in
tomato (Pino-Nunes et al., 2009). In addition, pollen viability has
been considered as an indicator of fertility for mutagenesis
programs of maize (Kumar and Rai, 2007) and several species of
legumes (Ashok et al., 2009); however, this method has never been
used to improve mutant populations.
The association of pollen viability with fruit seed production
allows the development of an early test to increase the productive
efficiency of mutant M2 lines by an initial preselection for fertility
in M1 plants. Early selection of mutant plants of Cucurbita pepo with
medium and high levels of pollen viability allows the screening of
more plants in less time and significantly increases the incidence of
fertile M1 lines, 21 and 40% for the treatments with 40 mM and
65 mM EMS, respectively.
Observations on several plant taxa differ regarding the potential
effect of fertility selection on the increase of mutation load. On the
one hand, the results of phenotypic evaluations of fertile lines of
EMS-treated plants in species such as the tomato (Hildering and van
der Veen, 1966) and barley (Gaul, 1958) support a model in which
completely fertile M1 plants yield mutant lines that possess the
same frequency of morphological mutations as those with
disturbed fertility. On the other hand, the induced mutations per
M2/M3 lines in Arabidopsis thaliana increased when derived from
M1 plants with lower seed fertility (Martín et al., 2009) as long as
mutation saturation allowed the viability of sexual reproduction.
Whether the mutation load is variable in mutant lines of C. pepo
selected based on pollen viability is a parameter that must be
clarified in order to transfer this method to many other species and
constitutes the next challenge in this mutant population when the
TILLING procedure has been established.
Recently, some specific genomics and transcriptomics tools,
which significantly support the breeding of this species, have
been developed for the ‘MU-CU16’ zucchini (Blanca et al., 2011;
Esteras et al., 2012). The addition of new induced-mutation
resources and the future development of a TILLING platform can
be expected to contribute another step toward expediting
zucchini breeding.
Acknowledgements
This work was supported by the project RTA2011-00044-C02-
01/02 from the Spanish Institute of Agronomy Research INIA
(Instituto Nacional de Investigación y Tecnología Agraría y
Almentaría). The authors thank Dr. M. Tellez for the design and
application of statistical methods. We are also grateful to the
Andalusian Institute of Agronomy Research IFAPA for the grant
provided to NVD.
 N. Vicente-Dólera et al. / Scientia Horticulturae 169 (2014) 14–19
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