Cucurbitaceae '96/EUCARPIA VI
"Cucurbits Towards 2000"
Biotechnology & Physiology
Expression of Agamous-like Genes in Male and Female Flowers of
Naomi Rosenman, Anat Kahana, Yu Xiang and Rafael Perl-Treves
Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900,
Cucumis sativus is a monoecious species in which sex
expression was extensively studied. Our laboratory at Bar-Ilan
is interested in the molecular basis of sex-expression in
cucumber. In this report we provide initial data on the cloning
and characterization of a family of cucumber genes that are
homologous to the Arabidopsis gene Agamous, that
specifies the identity of reproductive organs in bisexual
flowers. Considering unisexual flowers, it is very interesting
to examine the possible role of such organ-identity genes in the
selective development of one whorl of reproductive organs, while
the other is arrested.
We have prepared floral bud cDNA libraries from male and female
buds of an early developmental stage. The Agamous cDNA
from Arabidopsis (provided by Prof. Meyerowitz, Caltech)
was applied at moderate stringency to probe these libraries.
Three positive clones were selected for further study. The
clones appear closely homologous to each other and to the
Arabidopsis Agamous gene. The homology extends
also out of the MADS and K-box regions - proving that we cloned
true members of the Agamous sub-family within the MADS-
gene superfamily. When using the 3' half of each clone, gene-
specific bands are detected on cucumber genomic blots, in
addition to some bands that are shared by the other clones.
Organ-specific patterns of expression in male and female cucumber
flowers were observed by Northern analysis, and differed between
the clones. Attempts to clone other sex-specific genes by other
approaches, such as differential display, are under way in our
Genetic Map Construction and Map Merging in Cucumber (Cucumis
J. Staub, F. Serquen and J. Bacher
USDA, ARS, Vegetable Crops Research Unit Horticulture Department,
University of Wisconsin, Madison, WI (USA)
Abstract. The United States Department of Agriculture
cucumber (Cucumis sativus L.) improvement project
(USDA-CIP) is committed to germplasm enhancement by the
incorporation of unique genes (e.g., those conditioning multiple
lateral branching habit, sequential fruiting and disease
resistance) from exotic backgrounds (e.g., Cucumis sativus
var. hardwickii (R.) Aelf.) and unadapted Cucumis
sativus into North American-adapted processing cucumber
lines. The USDA-CIP is characterizing molecular markers
[isozyme, RFLP (restriction fragment length polymorphisms) and
RAPD (random amplified polymorphic DNA)] in cucumber for
germplasm evaluation and enhancement. Molecular markers have
potential for increasing the efficiency of breeding programs.
Genetic maps exist which possess valuable genetic information.
However, their value is limited because, individually, they do
not provide enough information for effective use in a cucumber
improvement program. If the information on all maps could be
merged into a unified map, then the potential utility of the
linked markers would be dramatically increased. The purpose of
this paper is to examine marker type, level of polymorphism, the
characterization of quantitative trait loci (QTLs) and map
construction in cucumber. The level of polymorphism in cucumber
is relatively low (~0.2 to 10%) and clustering of molecular
markers on genetic maps occurs. QTL analysis revealed genomic
regions associated with important yield and quality traits.
Nevertheless, the USDA-CIP has identified 70 RFLP and 80
RAPD markers in two plant populations and has determined their
spatial arrangement by constructing two genetic maps. The
potential utility of markers on these maps has been increased by
merging these maps.
Length Polymorphism and Homology of Microsatellites in Several
Y. Danin-Poleg1, G. Tzuri1, Z. Karchi1,
P.B. Cregan2 and N. Katzir1
1Department of Vegetable Crops, Agricultural
Research Organization, Northern Research Center, Newe Ya'ar,
P.O.B. 90000 Haifa 31900, Israel, and 2USDA-ARS, BARC-
West, Beltsville, MD 20705, USA
Abstract. The objectives of this research were to assess
(a) the degree of Simple Sequence Repeats (SSR) DNA length
polymorphism in melon (Cucumis melo L.) and cucumber
(C. sativus L.) and (b) the possibility of utilizing SSRs
flanking primers from a single species in other species of
Cucumis. Thirteen SSRs were tested in this study: eight
melon (CT/GA)n SSRs were isolated from a genomic
library; one melon and four cucumber SSRs were detected through a
DNA sequence databases search. Ten of the 13 SSRs tested (77%)
detected polymorphism in a sample of eight melon varieties. Gene
diversity values obtained with SSRs in melon were high (0.22-
0.83) with two to six alleles for each SSR in a sample of eight
varieties. Six of the 13 tested SSRs (46%) detected polymorphism
among 11 cucumber genotypes, with two to five alleles for each
SSR, correlating with gene diversity values of between 0.18 and
0.72. Codominant segregation of the alleles among F2
progeny was demonstrated for each of six SSR tested loci. The
possibility of using the same microsatellite flanking primers in
more than one species was proven.
Random Amplified Polymorphic DNAs (RAPDs) as Markers to
Determining Genetic Relationships Among Cuumis melo L.
E. García-Rodríguez1, I.
Alvarez2 and R. Lozano1
1Departamento de Biologia Aplicada (Unidad de
Genetica), E. Politecnica Superior, Universidad de Almeria, 04120
Almeria, Spain, and 2Centro de Investigacion, S & G
Semillas, S.A. 04738 El Ejido (Almeria), Spain
Genetic variability existing in Cucumis melo L. has been
previously examined through several studies. Isozymic
variability was found to be very low, while RFLPs detected enough
polymorphism to differenciate varietal groups, but not to
discriminate among lines belonging to a given group. The
analysis of regions of the C. melo genome revealed very
few variations at the DNA sequence level, which could account for
the paucity of RFLPs markers.
Nevertheless, previous studies have shown that melon genome
contains a high level of repetitive DNA sequences. Moreover, it
is well known the ability of RAPD markers to detect polymorphisms
in these DNA regions, what makes them useful to detect
intravarietal variations which can not be identified by isozyme
or RFLP analysis. Among the objectives of this study were to
analyze the applicability of RAPDs to detect variability in
Cucumis melo, particularly at the intravarietal level, and
to determine the genetic relationships existing among different
cultivars of this species.
The genotypes conforming the different groups are representative
of the germplasm of the cultivated melon in Spain and other
European countries. After screening of 80 primers, an average of
5-6 bands were clearly amplified and out of the 107 amplification
products generated by polymorphic primers (19 of them), 39 showed
scorable polymorphisms that allowed for the assessment of the
genetic distance values. These ranged from 0.034 to 0.240
suggesting that, compared to others species such as Lens (0.324
to 0.630), there is relatively few variability in Cucumis
melo. Despite the diversity ofthe material included in our
analysis, the number of primers employed detected enough
polymorphism to cluster together genotypes included in the same
varietal type. Charentais and Spanish types (yellow and green or
toad skin) were clustered together and separated from the Galia
type. At the intravarietal level, our results concerning to the
genetic relationships among Galia and Charentais types are in
agreement with the pedigree data known from agronomical
observations, suggesting that RAPDs are better suited than to
RFLPs in their capacity to detect intravarietal polymorphism in
Control of Melon Ripening by Expressing an Antisense ACC Oxidase
M. Guis, R. Botondi, R. Ayub, M. Ben Amor, P. Guillen, A.
Latche, M. Bouzayen and J-C Pech
ENSAT, Laboratoire Ethylene et maturation des fruits, 145
Avenue de Muret, 31075 Toulouse Cedex (E-mail:
Cantaloup charentais, the most cultivated melon type in France
exhibits very good organoleptic traits but has a very poor
storage capacity due to a sharp climacteric phase associated with
a very high ethylene production rate. Genetic manipulation of
ethylene production represents a suitable alternative for
improving shelf-life of this type of fruit. We generated melons
harbouring an antisense gene encoding ACC oxidase, an enzyme
involved in the last step of ethylene biosynthesis. Transgenic
melons exhibited reduced capacity to produce ethylene (less than
1% of control untransformed fruit at the climacteric peak). As a
consequence ripening was strongly inhibited both on and off the
vine (1). Analysis of the transgenic melons indicated that the
ripening process includes ethylene-dependent (aroma volatiles
production, chlorophyll and cell wall degradation, pigmentation
of the rind, activation of peduncular abscission zone) and
ethylene-independent pathways (coloration of the flesh). As
antisense fruit can be kept on the vine for longer, they can be
allowed to accumulate higher amounts of soluble sugars, thus
reaching better sensory quality than control untransformed fruit.
The antisense phenotype could be reversed by exogenous ethylene
Ayub, R., et al. 1996. Expression of ACC-oxidase antisense gene inhibits
ripening of cantaloupe melons. Nature Biotechnology 14:862-866.
Isolation and Fusion of Cucumis sativus and
Cucumis melo Protoplasts
M. Fellner1, P. Binarová1 and A.
1Institute of Experimental Botany, Academy of
Sciences of the Czech Republic, 772 00 Olomouc, Czech
Republic, and 2Palacký University, Faculty
of Natural Sciences, Department of Botany, 772 36 Olomouc-
Holice, Czech Republic
Abstract. The aim of this work was to transfer genes
responsible for resistance to cucumber downy mildew
(Pseudoperonospora cubensis) from C. melo to
C. sativus. The crossing barrier resulting from
their different chromosome number may be overcome by somatic
hybridization via protoplast fusion.
A set of C. sativus cvs. Bílské nakladacky,
Admira F1 and Pálava originated from the
Czech Republic. The C. melo genotypes PI 124111, PI
124112, PI 200819, PI 234607, PI 321005, MR-1 and CGN 2365
were provided by Dr. M. Pitrat (Montfavet, France), Dr. M.P.
Widrlechner (Ames, USA) and Mr. S. Zijlstra (Wageningen, The
Hypocotyls, cotyledons and true leaves from 4-6 day old
C. sativus and C. melo seedlings grown in
sterile conditions were cut into small segments and placed
into Erlenmeyer flasks on the solid initiation MS media with
growth regulators (2,4D, NAA, BAP, kinetin) and after ca two
weeks, the explants were transferred from initiation to
multiplication MS media without regulators or added with NAA
and BAP. Cell suspensions were initiated from selected
friable calli. One-week old cell suspensions were filtered
to remove large clumps of callus and then further cultured.
The protoplast isolation was achieved using enzyme
solutions. Protoplasts were isolated from embryogenic
suspension culture and from hypocotyls of 7 days old
seedling C. sativus cv. Bílské and from C. melo genotypes PI 124111 or PI 124112. Fusion of
protoplasts with 21% polyethylene glycol in drops of culture
medium was followed by washing in membrane stabilizing
medium (0.4M mannitol with 100 mM CaCl2). After
fusion, protoplasts were cultured: a) in liquid medium MS
2/2, b) in medium HS 2/2 solidified with 1.8% of agarose
(Sea Plaque), c) as the discs of protoplasts embedded in
agarose cultured in liquid MS 2/2 medium.
Vigorously growing callus cultures and cell suspensions were
produced from cotyledons and hypocotyls of C. sativus
and C. melo on several combinations of initiative and
multiplicative culture media. The calli derived from
hypocotyls of C. sativus reached a larger size when
grown in the presence of 2,4-D for 7 days. Calli from C.
melo were more heterogeneous in colour and structure in
comparison with C. sativus. Cotyledons of both
species were shown to be more responsive to variation in
culture media in comparison with hypocotyls. A combination
of growth regulators such as 2,4-D and BAP or kinetin seems
to be necessary for the formation and differentiation of
calli from C. sativus and C. melo explants.
Hypocotyl-derived calli and cell suspensions generally
provided higher number of protoplasts which were isolated by
treatment with nine enzymatic solutions. The maximal yield
of protoplasts was obtained from the cell suspension culture
of C. sativus cv. Bílské which was cultured
in MS 2/2 medium with light, using the BC enzymatic solution
(1.2% cellulase Onozuka R-10, 1.2% Macerozyme R-10, 0.3%
Driselase, 3 mM MES, O.1M glycine, CPW salts, mannitol) from
calli by using a KFK-I-CS enzymatic solution (2% cellulase
P-25, 0.4% Macerozyme R-10, 2% pectinase, 0.9%
CaCl2, mannitol). The optimal digestion time was
17 hours. Highest protoplast yields in C. melo were
obtained from the cell suspension of genotype PI 124112
cultured in MS 2/2 medium in light and from calli of
genotype PI 234607 by using the BC enzymatic solution. The
optimal digestion time was 8 hours. Protoplast culture
experiments were carried out with protoplasts of C.
melo PI 124112. After 24 hours, 19.6% of protoplasts
showed the regeneration of cell wall. Two days later, the
formation of small cell clusters was evident. Heterologic
fusion of protoplasts (C. sativus x C. melo)
was achieved with 21% polyethylene glycol. In fusion
products (C. sativus cv. Bílské x C.
melo PI 124111 and/or PI 124112) cell wall regeneration,
and sporadically first cell divisions, were observed.
Formation of microcolonies was recognized rarely,
regenerated plants were not obtained.
Bacterial Infection-like Lesions on Parthenocarpic Cucumber
Fruits During Ripening
Vegetable Crops Research Institute, Kecskémet (Hungary)
Introduction. Pseudomonas syringae pv.
lachrymans has been an important pathogen on field
cucumbers under Hungary's climatic conditions. Its presence has
especially importance on cucumber fields where seed production is
going on because of the seed-borne nature of this pathogen.
Those seed-stocks that are carrying one percentage of infected
seeds are excluded from the market by Hungarian rules. This is
the reason why Pseudomonas syringae pv. lachrymans
is inspected so strictly on seed producer cucumber fields.
Materials and Methods. Between 1978-1980 a number of
parthenocarpic cucumber hybrids (Colet, Marbel, Andrea, etc.),
varieties and lines appeared on our fields and breeding plots.
When we saw their fruits turning into ripening we were shocked
seeing how strong the bacterial infestation has developed on
them. The deceptive spots have been yellowish green or yellow,
3-8 mm in diameter with uncertain margin. Inside the spots the
skin was hypertonically puffy, uneven, having an oily glitter.
They were developing in a great number usually before the fruit
skin changes its green color into whitish or pale-yellow
corresponding to the ripening process.
In order to be sure of the infective agent we made isolations
from the characterized spots using the standard methodology of
this procedure (1).
Results. In the first year (1978) near a hundred
isolations were made and examined. In the next two years further
80 and 50 isolations but none of them yielded the suspected
pathogen P. syringae pv. lachrymans bacterium.
Studying the basical literature (2,3,4,5) we have not found
reference to that kind of change of fruit skin in parthenocarpic
During the time has passed since the beginning of our study we
ascertained that the described disorder of the ripening cucumber
fruit skin is a characteristic feature of parthenocarpic cucumber
lines and varieties everywhere and at any time in Hungary. In
progenies of crosses made with parthenocarpic lines only the
parthenocarpic entities are carrying the disorder. Summing up it
can be stated that the described disorder is a plant-
physiological feature (not infectious) and is inherited with the
parthenocarpy in the cucumber. It is not easy but highly
important to distinguish between symptoms of apparent similarity
yet of different origin, when surveying seed production fields
checking the presence of P. syringae pv.
Király, Z. (ed). 1974. Methods in plant pathology. Budapest
Akadémiai K. p. 509
Pike, L.M. and C.E. Peterson. 1969. Inheritance of
parthenocarpy in cucumber (Cucumis sativus L.) Euphytica
Ponti, O.M.B. de. 1976. Breeding parthenocarpic pickling
cucumbers (Cucumis sativus L.): Necessity, genetical
possibilities, environmental influences and selection criteria.
Robinson, R.W., H.M. Munger, T.W. Whitaker and G.W. Bohn. 1976.
Genes of the Cucurbitaceae. HortScience 11:554-569.
Rudich, J., L.R. Baker and H.M. Seli. 1977. Parthenocarpy in
Cucumis sativus L. as affected by genetic parthenocarpy,
thermophotoperiod, and femaleness. J. Amer. Soc. Hort. Sci.