Papers by Sergio Lucretti
Plant Breeding, 1988
Sixteen inbred lines and one hybrid of manse were tested for their capability of somatic embryoge... more Sixteen inbred lines and one hybrid of manse were tested for their capability of somatic embryogenesis, and fully developed plants could be regenerated, from ten inbred, lines. The highest frequency of plant regeneration was expressed in the inbred line CHI 31, and when this line was crossed with a recalcitrant, non-regenerating line, the F1 and BC hybrids were regenerable. The results of reciprocal crosses demonstrated that dominant nuclear genes and cytoplasmic factors are primarily responsible for the heritable determination of embryogenic callus proliferation and in vitro regeneration of maize plants.Somaclonal and radiation-induced variability was studied in maize to assess their nature and potential contribution to plant breeding., The inbred line CHI 31 possessing a high in vitro capacity of somatic embryo formation was used as experiments.] material. CHI 31 plants were selfed and twelve-day old zygotic embryos irradiated with 60Co gamma radiation in situ. Mature caryopses were harvested and assigned as M1 material. In another series, immature zygotic embryos (size 1.2—1.5 mm) were cultured in vitro on N-6 medium supplemented with 2,4-D (2.5 μM), and somatic embryos regenerated into plants; these were transplanted into soil and self-pollinated. Regenerants from non-irradiated cultures were grown as R1 generation, while regenerants from irradiated explants were considered as M1R1 generation.The genetic variability was evaluated in the M2, R2 and M2R2 generations, respectively, and compared with a non-treated seed control. Irradiation induced a variety of chlorophyll and morphological variants segregating in the M; generation; however, the frequency of deviant types obtained in the R: generation (somaclonal variation) was significantly exceeding the one derived from the M2 populations. The combination of expert irradiation and in vitro regeneration was most effective for the manifestation of chlorophyll and morphological o if types in the M2R2 generation, and increased drastically the frequency of early flowering variants. Differences in the segregation patterns of mutant phenotypes amonsister somaclones in the R3 and M3R3 generations indicate a different genetic basis, of plants originating from the same explant. This phenomenon suggests a mutational sectoring of the callus during culture. Radiation induced and somaclonal variation exerted a similar spectrum of chlorophyll and morphological deviants.
Physiologia Plantarum, 1992
Dolezel, J., Sgorbati, S. and Lucretti. S. 1992 . Comparison of three DNA fluorochromes for flow ... more Dolezel, J., Sgorbati, S. and Lucretti. S. 1992 . Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants.-Physiol. Plant. 85:· 625-631.
Plant Cell Reports, 2002
Somatic hybrids between Redblush and Duncan grapefruit (Citrus paradisi Macfadyen) with Avana, Ta... more Somatic hybrids between Redblush and Duncan grapefruit (Citrus paradisi Macfadyen) with Avana, Tardivo di Ciaculli mandarin (C. deliciosa Tenore) and Fortune mandarin (C. reticulata Blanco) were obtained by symmetric protoplast fusion. Isozyme banding pattern analysis and flow cytometry were used for early screening. DNA of the hybrids was then extracted and subjected to inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) using different primers anchored at the 5′ and 3′ ends. PCR products were resolved on 1.5% agarose gels and stained with ethidium bromide. All the bands from the parental cultivars were present in the somatic hybrids. The usefulness and efficiency of the ISSR-PCR method as a screening technique for Citrus somatic hybrids are discussed.
Scientia Horticulturae, 2007
Eighty-six triploid Citrus plants were recovered from interploid crosses between a natural tetrap... more Eighty-six triploid Citrus plants were recovered from interploid crosses between a natural tetraploid selection of the tangerine 'Dancy' (Citrus reticulata Blanco, cultigroup 'Tangerine'), used as the pollen parent, and two seedy selections of diploid mandarins (C. reticulata Blanco) as well as one clementine (Citrus clementina Hort. ex Tan.): 'Fortune' mandarin, 'Wilking' mandarin and 'Monreal' clementine. Flow cytometric analysis was used for screening the triploid plantlets and the ISSR-PCR technique was used to characterize the obtained triploids through a double approach, confirming the hybrid nature of the offspring and allowing the analysis of the genetic pool obtained. Selection among triploid genotypes generated from elite seedy parents will give us a better chance to obtain superior mandarin cultivars characterized by true seedlessness. #
Theoretical and Applied Genetics, 1996
A high-yield method for the isolation of intact nuclei and chromosomes in suspension from a varia... more A high-yield method for the isolation of intact nuclei and chromosomes in suspension from a variable number of pea root tips (1–10) has been developed. This procedure is based on a two-step cell-cycle synchronization of root-tip meristems to obtain a high mitotic index, followed by formaldehyde fixation and mechanical isolation of chromosomes and nuclei by homogenization. In the explant, up to 50% of metaphases were induced through a synchronization of the cell cycle at the G1/S interface with hydroxyurea (1.25 mM), followed, after a 3-h release, by a block in metaphase with amiprophos-methyl (10 μM). The quality and quantity of nuclei and chromosomes were related to the extent of the fixation. Best results were obtained after a 30-min fixation with 2% and 4% formaldehyde for nuclei and chromosomes, respectively. The method described here allowed the isolation of nuclei and chromosomes, even from a single root tip, with a yield of 1×105/root and 1.4×105/root, respectively. Isolated suspensions were suitable for flow cytometric analysis and sorting and PRINS labelling with a rDNA probe.
Chromosome Research, 1993
Chromosomes from reconstructed field bean (Vicia faba L.) karyotypes were flow-sorted and the DNA... more Chromosomes from reconstructed field bean (Vicia faba L.) karyotypes were flow-sorted and the DNA was used for the physical localization of seed storage and nonstorage (USP) protein genes using PCR with sequence specific primers. The data were confirmed and refined by using DNA of microisolated chromosomes of other karyotypes as the target for PCR. The specificity of the PCR products was proved by restrictase digestion into fragments of predicted length or by reamplification using ‘nested’ primers. The genes are located within defined regions of chromosome I (USP=unknown seedprotein genes), II (vicilin genes, legumin B3 genes), III (legumin B4 genes), IV (pseudogenes ψ1) and V (legumin A genes and pseudogenes ψ1). Except for the pseudogene derived from the sequence of legumin B4 gene, all members of each gene family are located in one chromosome region exclusively. This approach proved to be useful for localizing genes that cannot be mapped genetically (due to the lack of allelic variants) and might be applied to integrate physical and genetic maps.
Journal of Tissue Culture Methods, 1999
The analysis of structure and metabolism of a cell at a defined phase of cell cycle is often diff... more The analysis of structure and metabolism of a cell at a defined phase of cell cycle is often difficult because cell cycle progression in somatic tissues is asynchronous and only a fraction of cells are cycling. An elegant solution to obtain populations of cells enriched for single stage of the cell cycle is to impose the synchrony artificially. Different systems have been used to obtain synchronized populations of plant cells, including suspension-cultured cells, leaf mesophyll protoplasts and root tip meristems. Root tips have been frequently used in a variety of studies ranging from chromosome analysis to cell cycle and its regulation. Seedlings with actively growing roots may be obtained in most plant species, they are easy to handle, the experimental system is well defined, reproducible and can be easily modified for different species. This paper describes a protocol for cell cycle synchronization in root tips of Vicia faba, which is based on the use of DNA synthesis inhibitor hydroxyurea [18]. Modifications of the protocol for Pisum sativum, Medicago sativa, Hordeum vulgare, Secale cereale, Triticum aestivum, and Zea mays are also given. Flow cytometric data indicate that about 90% of root tip cells are synchronized. On average, mitotic indices exceeding 50% are obtained with the method. Synchronized cells may be accumulated at metaphase using a mitotic spindle inhibitor to achieve metaphase indices exceeding 50%.
Euphytica, 2010
We report the accurate determination of the allelic configurations of a total of eight new citrus... more We report the accurate determination of the allelic configurations of a total of eight new citrus tetraploid hybrids by means of SSR analysis, coupled with capillary electrophoresis, and PCR based dosage effects. Tetraploid hybrids were spontaneously obtained from different interploid crosses (2x × 4x) between diploid ‘Femminello’ lemon and the allotetraploid somatic hybrid (2n = 4x = 36) ‘Key’ lime + ‘Valencia’ orange, and between diploid ‘Wilking’ and ‘Fortune’ mandarins and an autotetraploid ‘Dancy’ mandarin (2n = 4x = 36). To understand the opportunity to employ them in further backcross programs, the cytological mechanisms underlying their ploidy level were unambiguously determined using six SSR primers. PCR conditions were optimized and skewness in template/product ratios were verified. Tetraploid allelic configurations were determined from PCR based dosage effects using electropherogram peak heights to estimate the copy number per allele. In all the tetraploid hybrids we found out that diploginy (2n eggs) has occurred, contributing the extra haploid genome in the tetraploids. According to the marker genotypes, it was further inferred that the 2n eggs in ‘Femminello’ lemon resulted from first division restitution (FDR), while in ‘Wilking’ and ‘Fortune’ mandarins 2n eggs occurred in second division restitution (SDR). These new genotypes, with their improved genetic female background, can be therefore considered very valuable in our citrus genetic improvement program as pollen donors in backcrosses suitable to eliminate negative traits.
Planta, 1992
A new method is described for the isolation of large quantities of Vicia faba metaphase chromosom... more A new method is described for the isolation of large quantities of Vicia faba metaphase chromosomes. Roots were treated with 2.5 mM hydroxyurea for 18 h to accumulate meristem tip cells at the G1/S interface. After release from the block, the cells re-entered the cell cycle with a high degree of synchrony. A treatment with 2.5 μM amiprophos-methyl (APM) was used to accumulate mitotic cells in metaphase. The highest metaphase index (53.9%) was achieved when, 6 h after the release from the hydroxyurea block, the roots were exposed to APM for 4 h. The chromosomes were released from formaldehyde-fixed root tips by chopping with a scalpel in LB01 lysis buffer. Both the quality and the quantity of isolated chromosomes, examined microscopically and by flow cytometry, depended on the extent of the fixation. The best results were achieved after fixation with 6% formaldehyde for 30 min. Under these conditions, 1 · 106 chromosomes were routinely obtained from 30 root tips. The chromosomes were morphologically intact and suitable both for high-resolution chromosome studies and for flow-cytometric analysis and sorting. After the addition of hexylene glycol, the chromosome suspensions could be stored at 4° C for six months without any signs of deterioration.
Theoretical and Applied Genetics, 1995
Flow cytometric analysis has been performed on chromosomes isolated from formaldehyde-fixed root ... more Flow cytometric analysis has been performed on chromosomes isolated from formaldehyde-fixed root tips in a Vicia faba (2n = 12) line with a standard (wild-type) karyotype and in six V. faba translocation lines with reconstructed karyotypes. The resolution of individual chromosome types on histograms of chromosome fluorescence intensity (flow karyotypes) depended on the type of fluorochrome used for chromosome staining. The highest degree of resolution was achieved with 4′,6-diamidino-2-phenylindole (DAPI). The lower resolution obtained after staining with mithramycin A (MIT) and propidium iodide (PI) was probably due to the sensitivity of these stains to changes in chromatin structure induced by formaldehyde fixation. After the staining with DAPI, only 1 chromosome type could be discriminated in the line with a standard karyotype. In the translocation lines, the number of chromosome types resolved on flow karyotypes ranged from 2 in the G and the ACB lines to all (6) chromosome types in the EFK and EF lines. Refined flow karyotyping permitted the sorting of a total of 15 different chromosome types from five of the translocation lines. It is expected that flow sorting of chromosomes from reconstructed karyotypes will become a powerful tool in the study of nuclear genome organisation in V. faba.
Cytometry, 1997
In the present study, we report on the development of bivariate flow karyotyping in the legume br... more In the present study, we report on the development of bivariate flow karyotyping in the legume broad bean (Vicia faba). We optimised chromosome staining with 48,6-diamidino-2-phenylindole and mithramycin A and analysed chromosome suspensions prepared from a line with standard (wild-type) karyotype and from six translocation lines with reconstructed karyotypes. Chromosomes were isolated from formaldehyde-fixed root tips after cell cycle synchronisation, and their fluorescence was analysed with dual-laser flow cytometry after the staining. High-resolution bivariate flow karyotypes were obtained in all broad bean lines analysed. Compared with univariate analysis, the bivariate analysis permitted discrimination of more chromosome types. However, peaks corresponding to newly resolved chromosomes were rather closely spaced, which could have compromised the purity of sorted fractions. With only a few exceptions, chromosome peaks were in a straight line, suggesting only minor differences in the AT:GC ratio among the chromosomes. These results indicate the limited potential of bivariate flow cytometric analysis and sorting in broad bean. Cytometry 28:236-242, 1997. r 1997 Wiley-Liss, Inc.
Biologia Plantarum, 2002
An integrated approach has been developed for targeted retrieval of microsatellite markers from s... more An integrated approach has been developed for targeted retrieval of microsatellite markers from selected regions of the field bean (Vicia faba L.) genome. The procedure is based on a combination of advanced physical and genetic mapping techniques and includes the following steps: 1) flow-sorting of metaphase chromosomes, 2) construction of microsatellite-enriched chromosome-specific DNA libraries, 3) isolation of polymorphic microsatellite sequences from the libraries, 4) testing chromosome specificity of the microsatellites using polymerase chain reaction with purified fractions of individual chromosome types, and 5) integration of chromosome-specific markers into a genetic map. Several strategies for isolation of microsatellite clones were tested, including direct screening of non-enriched libraries with single or mixed probes and screening of the libraries after one or two rounds of enrichment. Finally, the usefulness of this approach was demonstrated by the retrieval of novel markers from a selected portion of the largest field been chromosome (No. 1).
Methods in Cell Biology, 1995
Critical Reviews in Plant Sciences, 1994
Methods in Cell Biology, 2001
Theoretical and Applied Genetics, 1996
Chromosome Research, 1996
Recombinant DNA libraries were constructed for seven chromosome types isolated from two transloca... more Recombinant DNA libraries were constructed for seven chromosome types isolated from two translocation lines of field bean (Vicia faba L.) with reconstructed karyotypes. The chromosomes were selected so that the set of libraries covers the wholeV. faba genome more than once. Individual chromosome types were highly purified by flow sorting, and their DNA was amplified by degenerate oligonucleotideprimed (DOP) polymerase chain reaction (PCR) and cloned into a plasmid vector. The choice of restriction site present in PCR primer and refinement of cloning protocol resulted in high cloning efficiency and allowed generation of libraries consisting of about 106 clones from 250 or 1000 sorted chromosomes. The insert size ranged between 50 and 2200 bp and the mean length estimated in individual libraries varied between 310 and 487 pb. Hybridization of cloned fragments with labelled genomic DNA showed that about 60% of inserts represented unique or low-copy sequences. The suitability of the libraries for genome mapping was demonstrated by isolation of clones containing microsatellite motifs.
Journal of Tissue Culture Methods, 1999
We describe a protocol for flow cytometry analysis of cell cycle in plants using indirect immunol... more We describe a protocol for flow cytometry analysis of cell cycle in plants using indirect immunolabelling staining and Vicia faba, Pisum sativum and Zea mays root tip cells as model systems. The protocol is based on simultaneous analysis of two fluorescent signals. The first, obtained after staining with propidium iodide, is used to quantify total nuclear DNA content. The second, obtained after indirect immunofluorescent staining of bromodeoxyuridine (BrdUrd), is used to quantify the amount of BrdUrd incorporated into nuclear DNA. In an attempt to standardize the procedure, the effects of various conditions for partial DNA denaturation using HCl, as well as of BrdUrd concentration and incorporation time on flow cytometry DNA / BrdUrd content analysis have been studied. Maximum BrdUrd-linked fluorescence was observed after a 30 min pulse with 10 μM BrdUrd and after DNA denaturation with 1.5 N HCl (final concentration) for 30 min at 25 °C. Under these conditions, DNA content histograms with relatively small coefficient of variation (< 4%, full peak) could be obtained. To avoid non-specific staining of cytoplasm and cell walls, the protocol involves the use of nuclei isolated from formaldehyde-fixed tissues. Fixed isolated nuclei are stable and may be stored in hexylene glycol 0.75 M at 4 °C for prolonged periods prior to actual staining and analysis.
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Papers by Sergio Lucretti