Marcello Buiatti

Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories

A number of PCR-based techniques can be used to detect polymorphisms in plants. For their wide-scale usage in germplasm characterisation and breeding it is important that these marker technologies can be exchanged between laboratories, which in turn requires that they can be standardised to yield reproducible results, so that direct collation and comparison of the data are possible. This article describes a network experiment involving several European laboratories, in which the reproducibility of three popular molecular marker techniques was examined: random-amplified fragment length polymorphism (RAPD), amplified fragment length polymorphism (AFLP) and sequence-tagged microsatellites (SSR). For each technique, an optimal system was chosen, which had been standardised and routinely used by one laboratory. This system (genetic screening package) was distributed to different participating laboratories in the network and the results obtained compared with those of the original sender. Different experiences were gained in this exchange experiment with the different techniques. RAPDs proved difficult to reproduce. For AFLPs, a single-band difference was observed in one track, whilst SSR alleles were amplified by all laboratories, but small differences in their sizing were obtained.

Comparative determination of some phytohormones in wild-type and genetically modified plants by gas chromatography–mass spectrometry and high-performance liquid chromatography–tandem mass spectrometry

The analytical performances of two optimized analytical methodologies used for the determination of auxins, cytokinins, and abscisic acid in plant samples were critically compared. Phytohormones were extracted from Nicotiana glauca samples using a modified Bieleski solvent and determined both by gas chromatography-mass spectrometry (GC-MS), after derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) on the Bieleski extract without any further treatment. HPLC-MS/MS gave better results in terms of higher coefficients of determination of the calibration curves, higher and more reproducible recoveries, lower limits of detection, faster sample preparation, and higher sample throughput. Thus, two sets of N. glauca and N. langsdorffii samples, both wild-type and genetically modified by inserting the glucocorticoid receptor (GR) gene encoding for the rat glucocorticoid receptor, were first characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and then analyzed by HPLC-MS/MS. Significant differences in the phytohormone content between the two sample sets were found and are very important in terms of understanding the mechanisms and effects on growth processes and the development of transgenic plants.

Orientation behaviour and heterozygosity of sandhopper populations in relation to stability of beach environments

Felicita Scapini,’ Marcello Buiatti,’ Elvira De Matthaeis’ and Marco Mattoccia3 ‘Dipartimento di Biologia animale e Genetica, Universitri, via Romana 17, 50125 Firenze, Italy *Dipartimento di Biologia animale e dell’Uomo, Universitti “La Sapienza”, viale dell’Universitci 32, 00185 Roma, Italy 3Dipartimento di Biologia, Tor Vergata, II Universitd di Roma, via 0. Raimondo, 00173 Roma, Italy Key words: Amphipoda; behaviour; orientation; heterozygosity. Introduction The talitrid amphipod Talitrus saltator (Montagu) is a common inhabitant of sandy beaches, where it lives its whole life cycle, avoiding both the arid environment inland and submersion in sea water. Orientation towards the sea is critical for fitness, because good orientation is needed to avoid death by dehydration. During the night sandhoppers from Mediterranean populations migrate inland to feed, during the day they remain burrowed in moist sand at the water’s edge (Scapini et al., 1992). Orientation for zonal recovery occurs mainly by following a sun compass (see Pardi and Ercolini, 1986 for a review). This is of high survival value since these amphipods are not protected against dehydration by morphological structure and the narrow strip of moist sand at water’s edge is subjected to periodic and aperiodic shifting due to tides and storms. It has been shown that the sun compass is genetically determined. Differences among natural populations along the Italian Tyrrhenian and Adriatic shores are related to the orientation of the seashores where they live (Pardi, 1960; Pardi and Scapini, 1983; Scapini et al., 1985; Scapini and Buitatti, 1985; Scapini and Fasinella, 1990). However, the orientation tendency seems to be the result of a non-additive continuous interplay between individual learning and plasticity on one hand, and genetic variation on the population level on the other. It is through both processes that populations adapt to rapid environmental changes (Scapini et al., 1988; Ugolini and Scapini, 1988). Orientation, therefore, being a genetically determined character connected to fitness, for which the relative contributions of genetic and learning components 43

Combination of amplification and post-amplification strategies to improve optical DNA sensing.

The work evaluated a series of approaches to optimise detection of polymerase chain reaction (PCR) amplified DNA samples by an optical sensor based on surface plasmon resonance (SPR) (BiacoreX). The optimised procedure was based on an asymmetric PCR amplification system to amplify predominantly one DNA strand, containing the sequence complementary to a specific probe. The study moved into two directions, aiming to improve the analytical performance of SPR detection in PCR amplified products. One approach concerned the application of new strategies at the level of PCR, i.e. asymmetric PCR to obtain ssDNA amplified fragments containing the target capable of hybridisation with the immobilised complementary probe. The other strategy focused on the post-PCR amplification stage. Optimised denaturing conditions were applied to both symmetrically and asymmetrically amplified fragments. The effective combination of the two strategies allowed a rapid and specific hybridisation reaction. The developed method was successfully applied in the detection of genetically modified organisms.

Pleiotropic effect of the insertion of the Agrobacterium rhizogenes rolD gene in tomato (Lycopersicon esculentum Mill.)

Abstract The Agrobacterium rhizogenes rolD gene, coding for an ornithine cyclodeaminase involved in the biosynthesis of proline from ornithine, has been inserted in Lycopersicon esculentum cv Tondino with the aim of studying its effects on plant morphological characters including pathogen defense response. The analysis of plants transgenic for rolD did not show major morphological modifications. First generation transgenic plants however were found to flower earlier, and showed an increased number of inflorescences and higher fruit yield. Transformed plants were also analysed for parameters linked to pathogen defense response, i.e. ion leakage in the presence of the toxin produced by the fungus Fusarium oxysporum f. sp. lycopersici, and expression of the pathogenesis-related PR-1 gene. All the plants harbouring the rolD gene were shown to be more tolerant to the toxin in ion leakage experiments, with respect to the untransformed regenerated controls and the cv Tondino. PR-1 gene expression was quantitated by means of real-time PCR both at the basal level and after treatment with salicylic acid, an inducer of Systemic Acquired Resistance. In both cases the amount of PR-1 mRNA was higher in the transgenic plants. It seems therefore that the transformation of tomato plants with rolD could lead to an increased competence for defense response, as shown by toxin tolerance and increased expression of the Systemic Acquired Resistance marker gene PR-1. The results are finally discussed in view of their possible economic relevance.

Correlations between in vivo resistance to Fusarium and in vitro response to fungal elicitors and toxic substances in carnation.

SummaryWith the aim of ascertaining the existance of a correlation between in vivo resistance to Fusarium oxysporum f. sp. dianthi and in vitro response to fungal elicitors and toxic substances, phenylalanine ammonialyase and phytoalexin accumulation, on one hand, and resistance to culture filtrate, on the other, were assayed in “in vitro” cultures of three susceptible and four resistant Dianthus caryophyllus cultivars. Cultivars showing varying degrees of resistance in vivo either tolerated higher culture filtrate concentrations (‘Niki’) or showed high PAL activity and phytoalexin production when treated with Fusarium elicitor (‘Duca’), or responded positively to both treatments (‘Mei-Ling’, ‘Pulcino’). No such responses were shown in tissue cultures of susceptible cultivars. The differential response to the fungal elicitor seemed to be highly specific as genetic differences between cultivars were not observed in tissue cultures treated with other biotic (Phytophthora infestans) and abiotic (HgCl2) elicitors.

Randomness and multilevel interactions in biology

The dynamic instability of living systems and the “superposition” of different forms of randomness are viewed, in this paper, as components of the contingently changing, or even increasing, organization of life through ontogenesis or evolution. To this purpose, we first survey how classical and quantum physics define randomness differently. We then discuss why this requires, in our view, an enriched understanding of the effects of their concurrent presence in biological systems’ dynamics. Biological randomness is then presented not only as an essential component of the heterogeneous determination and intrinsic unpredictability proper to life phenomena, due to the nesting of, and interaction between many levels of organization, but also as a key component of its structural stability. We will note as well that increasing organization, while increasing “order”, induces growing disorder, not only by energy dispersal effects, but also by increasing variability and differentiation. Finally, we discuss the cooperation between diverse components in biological networks; this cooperation implies the presence of constraints due to the particular nature of bio-entanglement and bio-resonance, two notions to be reviewed and defined in the paper.

Genotype-hormone interaction in the induction of chromosome aberrations: Effect of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin on tissue cultures from nicotiana SPP. ☆

Abstract The effect of a synthetic auxin-like substance (2,4-D) and a synthetic cell division factor (kinetin) on the induction of chromosome aberrations was studied on tissue cultures of Nicotiana glauca and the tumorous amphidiploid hybrid Nicotiana glauca × Nicotiana langsdorffii . The aberration frequencies in normal Nicotiana glauca tissue were proportional to the length of time of culture in the presence of 2,4-D. Moreover, both 2,4-D and kinetin increased chromosome breakage in the habiatouated Nicotiana glauca tissue but not in the amphidiploid hybrid tissue. The data are discussed in terms of genotype-hormone equilibria in long-term development of plant tissue culture.

Phenotypic plasticity in sun orientation of sandhoppers

SummaryLittoral sandhoppers perform zonal orientation mainly by means of sun orientation, which in Mediterranean populations was shown to be inborn, associated with a genetically determined directional tendency adapted to the shoreline of each population. In case of changes of the shoreline the genetic heterogeneity of populations and learning ability may cooperate in providing adaptation both at the population and at the individual level. This hypothesis has been tested carrying on studies on the ontogenesis of the reactionnorm of this behavioural trait inTalitrus saltator (Montagu) (Crustacea: Amphipoda) of a Tyrrhenian population which from previous results shows a well adapted seaward orientation. Sun orientation was analysed away from the sea shore, both in laboratory-raised individuals of different ages and in individuals captured in the field, by repeated releases on dry flat sand or in a glass bowl. Results showed variability within and between individuals of directional tendency, which varies in the individual life depending on age and experience. Particularly, age seems to bring canalization as variability within individuals tends to decrease with age. In order to evaluate the role of experience, we trained individuals to direct themselves towards a direction different from that genetically determined. The majority of the individuals showed a learning ability in the training situation. The importance of phenotypic plasticity in the oriented behaviour of sandhoppers is discussed.

Construction of a new vector conferring methotrexate resistance in Nicotiana tabacum plants

A new binary vector encoding for Candida albicans dihydrofolate reductase (DFR1) has been constructed and used as a dominant selectable marker for plant transformation. Transgenic tobacco plants with an increased resistance to methotrexate (Mtx) were obtained by co-transformation of tobacco leaf discs with Agrobacterium tumefaciens strains carrying two new binary vectors: pTI20 and pTI18. Co-transformants of Nicotiana tabacum were directly selected for and rooted on medium containing both kanamycin (kan) and Mtx. Leaf discs of transgenic plants were assayed for capacity of regeneration at different Mtx concentrations. Analysis of transcripts was performed on total RNA extracted from two Mtx-resistant plants. The transgenic plants increased resistance to Mtx can be explained by the exceptionally low capacity of Mtx to bind C. albicans dihydrofolate reductase, accountable by the presence of two amino acid residues strategically important in Mtx binding.