1996 Cucurbitaceae EUCARPIA VI Cucurbits Towards 2000 (B&P)

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Section: Biotechnology & Physiology

Abstracts

Expression of Agamous-like Genes in Male and Female Flowers of Cucumber

Naomi Rosenman, Anat Kahana, Yu Xiang and Rafael Perl-Treves

Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

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 laboratory.

Genetic Map Construction and Map Merging in Cucumber (Cucumis sativus L.)

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 Cucurbitaceae Species

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. Genotypes

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 C. melo.

Control of Melon Ripening by Expressing an Antisense ACC Oxidase Gene

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: [email protected])

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 treatment.

Literature cited:

  1. 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. Lebeda²

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 Netherlands). 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

Hódosy, A.S.

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 cucumbers.

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. lachrymans.

Literature cited:

  1. Király, Z. (ed). 1974. Methods in plant pathology. Budapest Akadémiai K. p. 509
  2. Pike, L.M. and C.E. Peterson. 1969. Inheritance of parthenocarpy in cucumber (Cucumis sativus L.) Euphytica 18:101-105.
  3. Ponti, O.M.B. de. 1976. Breeding parthenocarpic pickling cucumbers (Cucumis sativus L.): Necessity, genetical possibilities, environmental influences and selection criteria. Euphytica 25:29-40.
  4. Robinson, R.W., H.M. Munger, T.W. Whitaker and G.W. Bohn. 1976. Genes of the Cucurbitaceae. HortScience 11:554-569.
  5. 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. 102:225-228.