Rocket: a Mediterranean crop for the world - Bioversity International

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36 ROCKET GENETIC RESOURCES NETWORK ']XSPSKMGEP WXYH] SR VSGOIX WTIGMIW F] QIERW SJ MQEKI EREP]WMW W]WXIQ S. Blangiforti and G. Venora Wheat Experimental Station for Sicily, Caltagirone, Italy Introduction Rocket is a crop that has been known for many centuries. Romans utilized it broadly as a vegetable and condiment plant. Although different species are referred to under the common name of rocket, the most common ones are those belonging to Eruca and Diplotaxis genera. These genera belong to the subtribe Brassicinae, which includes 10 genera, according to the classification made by Schulz (1919, 1923, 1936). The economic interest in rocket is growing. This is particularly due to the diffusion of ready-to-use salads (4th generation of vegetables) that extend the shelf life of rocket leaves and preserve their freshness and the typical scent. However, a good number of varieties to meet the large market demand are still lacking. To achieve this goal, it is necessary to proceed to a systematic collection of germplasm in different regions of the Mediterranean area, where rocket occurs spontaneously and where targeted crop genetic improvement would yield great benefits. With regard to genetic improvement, the role played by cytological studies is essential to contribute to a deeper characterization of the species and thus to a more sensible classification of their genetic resources. A direct impact of these studies is most evident in hybridization activities. It is known that Brassicaceae generally have chromosomes that are difficult to observe, owing to their very small size. This is also true of Eruca and Diplotaxis, whose chromosome number in many species is the only known cytological information, any other indication on the morphology being lacking (Warwick and Anderson 1993). Recently, the use of image analysis, also applied to the karyotyping of plant chromosomes in species with difficult chromosomes, has allowed the accomplishment of detailed karyotypes (Venora et al. 1991, 1995a, 1995b, 1995c; Ocampo et al. 1992; Venora and Saccardo 1993; Venora and Padulosi 1997). This paper reports on the use of this innovative technique for the karyotyping of some rocket species (Eruca and Diplotaxis) which have so far not been investigated for this purpose. Materials and methods Seeds of Eruca vesicaria subsp. sativa (Mill.) Thell., Eruca vesicaria subsp. pinnatifida (Desf.) Emb. and Maire and Diplotaxis tenuifolia (L.) DC. were kindly supplied by Prof. Gomez-Campo of the Universidad Politecnica de Madrid (ETSIA). Seeds were germinated on moist filter paper in Petri dishes kept in the dark at room temperature. For the analysis of the somatic chromosomes, young and turgid primary roots (0.5-1 cm long) were cut off and pretreated in a saturated water solution of 1,4 dichlorobenzene for 2 hours at 15°C. They were then fixed in ethanol-acetic acid (3:1) for 24 hours at 4°C, after a thorough rinsing in water. The staining process was performed following the Feulgen technique by hydrolyzing the material in 5N hydrochloridric acid at room temperature for 55 minutes. The stained roots were then squashed in 45% acetic acid and mounted in Entellan (Merk).
+)2)8-' 6)7396')7 &6))(-2+ %2( '908-:%8-32 The microscopic investigation was conducted by using a Zeiss Axioplan 2 microscope connected to an image analysis system KS400 Kontron, with dedicated to karyotyping software ’KSChromo’ that enables more reliable results than the traditional hand-made karyotyping procedure (Venora et al. 1991). Total chromosome length, as well as the length of short arms, long arms and satellites, were all measured with this computerized system. All the data obtained by each plate were loaded in the dedicated software ’Karyo 95’ for the better matching of chromosome couples, which was done automatically on the basis of each chromosome data (Pavone et al. 1995). The short/long arm ratio does not include the satellite. The nomenclature of Levan et al. (1964) was followed in classifying the chromosomes. With regard to chromosomal indices, the classification of Stebbins (1971), the TF% index (Huziwara 1962) and the Rec and Syi indices (Greilhuber and Speta 1976) were used. The classification of Stebbins (1971) is based on the relative frequency of chromosomes with a long arm ratio greater than 2 and on the ratio between the lengths of the longest and the shortest chromosomes in the complement. The TF% index is expressed by the ratio between the sum of the lengths of the short arms of individual chromosomes and the total length of the complement. The Rec index expresses the average of the ratios between the length of each chromosome and that of the longest one. The Syi indicates the ratio between the average length of the short arms and the average length of the long arms. Results and discussion Figure 1 shows a typical metaphase plate with 22 chromosomes. In Figure 2 are represented the idiograms of the haploid complement of Eruca vesicaria subsp. sativa, E. pinnatifida and D. tenuifolia, obtained by using the image analysis system and the software ’Karyo 95’. The 3 species’ chromosome number is 2n=22, which is in agreement with previous reports from Warwick and Anderson (1993). The asterisks indicate the dissimilar pairs between the two Eruca species; the greater differences concern the first pair and the ninth pair (in general such differences are attributable to chromosomal rearrangements due to pericentromeric inversions). The idiogram of D. tenuifolia shows a karyogram similar to Eruca, but each chromosome is longer, and the karyotype formula is different. The karyotypic data have also been used to speculate on the degree of karyotype evolution in each species (Fig. 3). On the basis of these preliminary results, it seems that the two Eruca species are relatively more evolved than Diplotaxis species, from a karyotypic point of view, while being very similar to each other. Additional work is, however, necessary to confirm these data, possibly by using a greater amount of seeds and good metaphase plates. This study has been useful in providing cytogenetic information on these little-studied species. It has also been particularly successful in karyotyping (with the use of image analysis system) such difficult chromosomes as those of Eruca and Diplotaxis.
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