SESSION III - GENETICS AND BREEDING OF NO FOOD PLANTS

ORAL COMMUNICATION

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S3a

EXPLOITING THE PLANT PROTEIN SECRETORY PATHWAY FOR PRODUCTION OF RECOMBINANT MOLECULES FOR PASSIVE AND ACTIVE IMMUNISATION

J. MA, D. CHARGELEGUE, P. DRAKE, P. OBREGON
 
St. George's Hospital Medical School, London SW17 0RE
julian.ma@kcl.ac.uk

One of the key advantages that plants offer for the expression of recombinant proteins is that they are higher eukaryotic organisms with an endomembrane system. Therefore they fold and assemble recombinant proteins using protein chaperones that are homologous to those in mammalian cells, and they perform post-translational modifications. 

Plants expressing IgG monoclonal antibody was first described in 1989. Since then, transgenic plants have proven to be extremely versatile, successfully used for antibody fragments, IgG and secretory IgA antibodies, the latter already having been taken through clinical trials. A considerable amount of work has also been performed on the expression of antigens in plants, particularly with a view to oral delivery. To date, three examples have been tested in humans - E. coli heat labile toxin, hepatitis B surface antigen and Norwalk virus capsid protein. 

Overall, proof of principle has been demonstrated for the production of recombinant pharmaceutical in plants. Plants would appear to represent one of the few systems capable of delivering complex glycoproteins in the quantities required for vaccines, particularly in developing countries. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S3b

THE CONTROL OF THE CHEMICAL PHENOTYPE IN CANNABIS SATIVA L.: GENETIC ANALYSIS AND MOLECULAR MARKERS  

G. MANDOLINO*, A. CARBONI*, D. PACIFICO*, P. RANALLI*, E. DE MEIJER**

*) Istituto Sperimentale per le Colture Industriali, Via di Corticella 133, 40128 Bologna (I)
**) GW Pharmaceuticals plc., Porton Down Science Park, Salisbury, Wiltshire SP4 0JQ (U.K.)

Cannabis, cannabinoids, THC-synthase, CBD-synthase, molecular markers

Cannabinoids are terpenophenolic compounds unique to the genus Cannabis. These secondary products are important for breeding and legal reasons; in fact, UE subsidies are issued only if the amount of the psychoactive cannabinoid delta-9-tetrahydrocannabinol (THC) in the fibre crops is certified to be lower than 0.2 % of the inflorescence dry matter. Besides, the plants containing high levels of this cannabinoid are considered illegal, and are strongly repressed by police actions. On the other hand, high percent of specific cannabinoids (and especially of the psychoactive ones) are desirable during the breeding for the production of pure lines for pharmaceutical purposes. Therefore, the type and amount of cannabinoids produced by the Cannabis plants, and the mechanisms of their inheritance are of major interest for the different and sometimes contrasting purposes of breeding for this ancient no food plant.  

The two most common cannabinoids found in Cannabis plants are delta-9-tetrahydrocannabinol and cannabidiol (CBD, non psychoactive). These two compounds are synthetized from the common precursor cannabigerol (CBG, non psychoactive) by the two enzymes THC- and CBD-synthase. Inbred lines were obtained, endowed with only one of the two main cannabinoids (THC or CBD). Hybrid F1 progenies were obtained, and several individual F1 plants were selfed to obtain F2 generations. All the  F1 and F2 plants were analysed by gas-chromatography to determine their cannabinoids profile. The results obtained support a model of determination of the chemotype by one single locus (B) with two codominant alleles, BD and BT, each probably coding for one of the two synthases involved in cannabinoid biosynthesis; the chemotype distribution in all the F2 examined fitted well the expected 1.2:1 ratio. A null allele, B0, is also postulated, explaining the occurrence of CBG-accumulating plants. Sequence-characterized markers were developed using the two F2 pure-chemotypes segregating groups in a Bulk Segregant Analysis. These markers show tight linkage with the chemotype in the progenies studied. Besides, sequence-specific markers for each of the two alleles involved in chemotype determination were developed; a multiplex assay was set up for the simultaneous recognition, in one single PCR reaction, of the genotype at the B locus (BT/BT, BD/BD or BT/BD). Such assay allows the identification at very early stages of the type of cannabinoid each plant will produce at maturity. It has been noted that mixed chemotype plants (either the F1 hybrids or the 50% of each F2 progeny) fall into two different classes, depending on their CBD/THC content ratio. These different ratios are stably inherited from F1 hybrids to F2 heterozygous they generate, and suggest the existence of different isoforms of THC- or CBD-synthases, with different efficiencies in competing for the precursor CBG, and presumably encoded by different BT and BD alleles. This hypothesis is presently being tested by sequencing the THC- and CBD-synthase genes of F2 plants showing such different ratios of the two main cannabinoids.

Within each chemotype (i.e. within each genotype at the B locus), the cannabinoid content is a typically quantitative trait, probably related with the density of the glandular trichomes, the sites of the cannabinoid synthesis. Therefore, the overall chemotype is the net result of both a mendelian determination at one single locus (B) and of quantitative and environmental factors affecting the amount of each cannabinoid synthetized by the plant. Both these factors must be taken into consideration for the practical exploitation of these class of secondary products in breeding programs.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S3c

TRITERPENOID SAPONINS FROM ASTER SEDIFOLIUS: CHEMICAL CHARACTERIZATION, ANTIFUNGAL ACTIVITY AND EXPRESSION OF 2,3-OXIDOSQUALENE CYCLASE GENE

M. CAMMARERI*, M. ZACCARDELLI**, G. COREA***, V. LANZOTTI****, A. ERRICO*, C. CONICELLA*****

*) Dept. of Soil, Plant and Environmental Sciences, University of Naples "Federico II", via Università 133, 80055 Portici-NA (Italy)
**) Research Institute for Industrial Crops - MiPAF - ss.18, n° 156, 84091 Battipaglia -SA (Italy)
***) Dept. of Natural Product Chemistry, University of Naples "Federico II", via D. Montesano 49-I, 80131 Napoli (Italy)
****) Dept. of Science and Technologies for Food, Environment and Microbiology, University of Molise, Via De Sanctis, 86100 Campobasso (Italy)
*****) CNR-IGV, Research Division Portici, via Università 133, 80055 Portici-NA (Italy)

triterpenoid saponins, Trichoderma viride, 2,3-oxidosqualene cyclase gene 

Plants elaborate a vast array of natural products, which can be interesting for pharmaceuticals and biopesticides. The saponins are glycosides occuring in more than one hundred plant families and in some marine animals. In particular, triterpene saponins are isoprenoidal natural products with remarkable structural varieties as well as notable biological activities. 

The triterpenes are biosynthesized from a common precursor: 2,3-oxidosqualene. The cyclization of 2,3-oxidosqualene into tetracyclic and pentacyclic carbon skeleton is catalyzed by enzymes termed 2,3-oxidosqualene cyclases (OSCs). They exhibit a highly similar amino acid sequences, such as QW and DCTAE motifs.

The material which has been utilized in the present work for gene cloning and new substances purification is coming from a germplasm collection of Aster spp. The research dealed with the following topics: 1. the chemical structure of triterpenoid saponins extracted from Aster sedifolius; 2. the analysis of their antifungal activity in plant; 3. the expression of 2,3-oxidosqualene cyclase gene involved in the biosynthetic pathway.  

Three novel substances isolated from aerial parts of Aster sedifolius were characterized as triterpenoid saponins with oleanetype skeleton. Anti-tumoral effect of these substances is in progress. Biological tests with leaf extracts against Trichoderma viride and Botrytis cinerea evidence inhibition of fungal mycelium growth. Expression studies have been performed by RT-PCR with specific primers which amplify  a fragment of 300 bp in 2,3-oxidosqualene cyclase gene of Aster sedifolius. The results evidence differential tissue expression between in leaf, flower and root.

POSTER

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.01

ISATIS TINCTORIA L. (WOAD): GENETIC RESOURCES OF A NEARLY FORGOTTEN SPECIES

G. SPATARO, P. TAVIANI, V. NEGRI

Dipartimento di Biologia Vegetale e Biotecnologie Agro-ambientali (DBVBA), Università degli Studi di Perugia

genetic resources, Isatis tinctoria, molecular marker

Isatis tinctoria was widely cultivated in Europe to produce indico, a natural blue dye; this crop was completely abandoned aside at the end of the eighteen century, when synthetic dyes came into use.

Nowadays, the interest in I. tinctoria is increasing: the market demand of natural dyes is rising, as these represent an alternative solution of synthetic ones.

Aim of this study was to evaluate different populations for:
1.	adaptability at different altitudes. The characters taken into account were: leaves colour, plant carriage and vigour, pre-flowering cases.
2.	the morphologic and physiologic diversity. The characters analyzed were: leaves colour and size, cases of hairness leaves, plant carriage and vigour, pre-flowering cases.
3.	the genetic diversity using molecular markers as AFLP (Amplified Fragment Length Polimorfism) on bulk DNA.

The three experiments revealed a high level of polymorphism for both the morphologic and the genetic side: the populations appear to be very different each other and, contrary to all expectations, the more interesting accessions are the oriental ones. The local and adapted population (Borgorose) seems not to be agronomically interesting because of the plant carriage and vigour, the leaves colour and for the presence of pre-flowering cases.

The results related to the agronomic characters (vigour in particular) suggest the possibility of reintroducing successfully the crop in areas quite different for their pedoclimatic conditions.

Besides a wide genetic variability among population was found suggesting that efficient breeding program could be carried out when would be necessary to create varieties of this species. The AFLP molecular markers revealed that, in spite of the high outcrossing and the important gene flow, the different selective pressures caused the genetic characterization of population in different habitats.

Moreover, the molecular analysis confirmed that the centre of origin of I. tinctoria is likely to be Central Asia as many other Cruciferae.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.02

STUDIES OF GENES INVOLVED IN THE METABOLIC STEPS OF INDIGO PRODUCTION IN ISATIS TINCTORIA

M. DURANTE*, M. MANZO*, R. BERNARDI*, F. CECCONI*, M. SALVINI**, L. ANGELINI***

*) Dipartimento di Biologia delle Piante Agrarie, Sezione di Genetica, Via Matteotti 1/B, 56124 Pisa
mdurante@agr.unipi.it, rbernard@agr.unipi.it
**) Scuola Normale Superiore di Pisa, Piazza dei Cavalieri 7, 56126 Pisa 
msalvini@agr.unipi.it 
***) Dipartimento di Agronomia e Gestione dell’Agro-ecosistema, Via S. Michele degli Scalzi 2, 56124 Pisa 
angelini@agr.unipi.it  

Indigo, Isatis tinctoria, tryptophanase gene

Plant stain utilisation is well documented from ancient times: British Picti represent a historical known case of a people using skin decoration with indigo extracted from the woad plant Isatis tinctoria L.

The present work aims at the genetic improvement of plants producing  the indigo stain and to the re-introduction of plants yielding high indigo. In this direction, we are interested to individuate the genes involved in the metabolic pathway of indigo biosynthesis in Isatis tinctoria L., for their use in assisted selection programs.

We have hypothesised candidates genes involved in the metabolic pathways and we have designed degenerated primers for the amplification and isolation of these genes. At the moment, we are studying the alfa-tryptophanase gene involved in the the first step of indican biosynthesis, the indigo precursor  normally stored in vacuoli, and the beta-D-glucosidase gene involved in indican hydrolysis leading to the release of  indoxyl groups and  indigo synthesis.

In this context, we have isolated plants with high indigo production,  we have amplified the beta-D-Glucosidase and the alfa-Tryptophanase genes. 
 
Moreover, the expression of these genes during different developmental stages is under study through analysis of mRNA biosynthesis. 

This work is carried out within the EC Spindigo Project.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.03

CANNABIS SATIVA: A GENETIC PLANT MODEL FOR INDUSTRIAL END USES

P. RANALLI, G. MANDOLINO, A. CARBONI, M. DI CANDILO

Istituto Sperimentale per le Colture Industriali, Via di Corticella 133, 40128 Bologna

multi-use crop, cannabinoids, sexual differentiation, industrial uses

New crops are required for specialist needs, including raw materials for the chemical and pharmaceutical industries (bioethanol from sugar crops, fuel from oil crops, energy from biomass production). As a result, the redeployment of surplus agricultural resources and a switch away from dependence on fossil resources have been attempted. The development of plants for specialised productivity needs an enhanced understanding of metabolic control both in terms of molecular and genetic mechanisms and the consequences of remodelling of metabolic pathways. Hemp is a multi-use crop able to provide fiber, cellulose, seeds and seed oil, cannabinoids and biomass. Textile, paper, composite, fuel, energy, and pharmaceutical products are developed from this raw material. Integrating many agroindustrial chains, Cannabis is considered a crop model in which insights into specific metabolic pathways and biosynthetic processes are valuable for improvement of the plant for all sets of industrial derivatives. The research on hemp, supported by a project funded by the Italian Ministry of Agriculture, dealt with the following tasks: 1) the recovering and maintenance of hemp germplasm accessions in a viable condition and their reproduction; new methods of hemp genetic resource evaluation based on DNA analysis were also performed; 2) the identification of molecular markers for relevant traits (male phenotype and THC genes) and their allocation in a saturated linkage map were performed. The identification of QTL linked to other important characters, such as fibre content, is an important task in progress. In detail, efforts were focused to gain insight into the genetic factors underlying the sexual differentiation and the expression of the most important cannabinoid categories stored in the plant. Cannabis is a dioecious species with unisexual flowers and sex determined by heteromorphic chromosomes (X and Y). The gene expression of male and female apices was compared by cDNA AFLP. Seven of the several polymorphic fragments have been identified to be differentially expressed which belong to nine different mRNAs induced to female apices. The results of BLAST analysis for these clones led to the identification of their putative coding function and to their possible roles in the earliest step of sexual differentiation. Furthermore, the two most relevant cannabinoids synthesized in Cannabis plants are delta-9-tetrahydrocannabinol (delta9THC, psycoactive) and cannabidiol (CBD, non psychoactive) both originated from the common precursor cannabigerol (CBG, non psychoactive) by two enzymes THC-and CBD-synthase. Genetic analysis of inbred lines, and of progenies F1 and F2 support a monogenic model controlling the chemotype: one single locus (B) with two codominant alleles, BD and BT, each probably encoding one of the two synthases involved in cannabinoid biosynthesis; the F2 generations fitted the expected 1:2:1 chemotype segregation ratio. Sequence-specific markers for each of the two alleles involved in chemotype determination were also developed. Another relevant target is to increase the seed oil content and composition of cannabis oil. To date, cloning of lipid-associated genes from Cannabis have not been reported. However, the availability of heterologous genes for direct introduction into hemp, or their availability to design primers for isolating the corresponding cannabis homologous by PCR-based techniques is relatively advanced. Finally, fiber improvement is assisted by metabolic pathway engineering accomplished in cotton for introducing modified alcaligene eutrophus genes encoding the enzymes acetacetyl-CoA reductase and polyhydroxyalkanoate synthase. These two enzymes, when produced in fibre cells along with endogenous B-ketothiolase, catalyse the polymerization of acetyl-CoA to polyhydroxybutyrate. The transgenic fibres exhibited better insulating properties. The isolation of other relevant genes for the remodelling of pathways is a diminishing hurdle thanks to the pace of DNA sequence analysis of small plant genomes and the proliferation of EST (expressed sequenced tag) libraries. The huge genetic diversity exhibited by hemp germplasm allows access to relevant genes for specific functions. The advances in hemp basic and applied research are elucidated in this review; there are elements to identify strategy for design modified plants with enhanced industrial end uses. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.04

Agronomic and molecular evaluation of Kentucky bluegrass germplasm collected in Italy

F.B. Bertoli*, R.C. Johnson**, B. Lucaroni*, M. Romani***, L. Russi*, E. Piano***, M. Falcinelli*

*) Di.B.V.B.A., University of Perugia, Perugia, Italy
**) USDA ,W.R.P.I.S., Washington State University, Pullman, USA
***) I.S.C.F., Lodi, Italy

Poa pratensis, turfgrass, breeding, AFLP

Kentucky bluegrass (Poa pratensis L.) is a forage and turf grass, widely cultivated in temperate climates throughout the world. Its facultative apomixis, highly polyploid and even aneuploid genome hamper traditional breeding strategies aimed at obtaining superior genotypes. On the other hand, the high versatility of the mode of reproduction, that spans from near obligate apomixis to out-crossing sexuality, and the retention of a pollen recognition system have created a high degree of genetic variation in nature. This variation can be exploited directly by singling out superior apomictic genotypes, thus shortening the development of  new cultivars. The collection and the evaluation of native germplasm, well adapted to different climates, could also be of great interest for a low input, sustainable turf management. Sixteen indigenous populations, collected in North and Central Italy, were evaluated for potential turf usage and seed production in two contrasting environments in Italy, the Po Valley (Lodi) and Central Italy (Perugia). Moreover, AFLP DNA markers have been used to fingerprint the germplasm in order to assess genetic variation between and within populations. The AFLP DNA showed a wide range of the similarity values, indicating high variation among the populations. Populations from North Italy and those from Central Italy were sharply distinguished by means of both molecular markers and agronomic traits. The research suggests that adaptation of Poa pratensis indigenous to Italy is dependent on site of origin associated with different environments. This information will enhance selection resources for developing Poa pratensis cultivars for Italian conditions.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.05

ANALYSIS OF SICILIAN GERMPLASM OF HELICRYSUM GENUS USING AFLP MARKERS

G. M. ABBATE*, C. AGRIMONTI**, A. SCIALABBA*, N. MARMIROLI**

*) Dipartimento di Scienze Botaniche, Università degli Studi di Palermo
**) Dipartimento di Scienze Ambientali, Università degli Studi di Parma

Helichrysum, taxonomy, molecular markers, AFLPs

Helichrysum  is a wild shrub that grows in arid areas, in stony ground, on scarps and it requires sun exposition. The drug, prepared from the characteristic gold capitulums, displays several well known pharmacological properties (antinflammatory, antiallergic, antipsoriasic) but at present it is only employed in cosmetic field.

This genus, that is widespread throughout Mediterranean area, in Sicily is represented by the following species: H.italicum; H.stoechas; H.nebrodense; H.hyblaeum; H.scandens; H.siculum; H.litoreum and H.rupestre with the varieties rupestre, messerii, pendulum and  errerae. For some taxa, mostly ascribable to the H. rupestre group, is difficult to establish the exact taxonomic position; this is due to the variability of distinctive morphologic traits, that involves a wide phytochemistry variability, too. 

The analysis of DNA sequences as molecular markers, is an efficient method to escort the traditional taxonomy. For this purpose, AFLP (Amplified Fragment Length Polymorphisms) have been reported as a suitable tool for analysis of genetic diversity in different species.

In this work AFLP were used to assess the taxonomic position of some populations of sicilian Helichrysum  whose classification is uncertain.

The populations were collected from several areas of Sicily and surrounding islands.
To store this material, the seeds were collected at “Banca del germoplasma” of “Dipartimento di Scienze Botaniche” of University of Palermo. Moreover, to propagate the populations collected, from each sample were prepared scions and and in vitro culture. Further, DNA was prepared from collected material to perform AFLP technology. The analysis of profiles obtained is still in progress. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.06

ESTIMATION OF GENETIC VARIATION AMONG WILD CARDOON POPULATIONS USING AFLP ANALYSIS

E. Portis*, G. mauromicale**, E. SABA***, L. quagliotti*, A. Acquadro*, c. comino*, S. Lanteri*

*) Di.Va.P.R.A. settore Genetica Agraria, Università di Torino
**) Dipartimento di Scienze Agronomiche, Agrochimiche e delle Produzioni Animali - sezione Scienze Agronomiche, Università di Catania
***) Dipartimento di Scienze agronomiche e Genetica vegetale agraria, Università di Sassari

Cynara cardunculus var. sylvestris, Genetic variability, AFLP, Sicily, Sardinia

Wild cardoon [Cynara cardunculus L. var. sylvestris (Lamk) Fiori] is a rustic and robust perennial plant with a characteristic rosette of large spiny leaves and branched flowering stems. It is native to the Mediterranean basin where it colonizes dry and uncultivated areas. Cytogenetic as well as isozyme studies show that wild cardoon is the ancestor of the cultivated forms C. cardunculus var. scolymus L. (artichoke) and C. cardunculus. var. altilis DC (cardoon), since they are fully cross-compatible and their F1 hybrids are fully fertile. 

Previous studies showed that wild cardoon can be exploited for the production of lignocellulosic biomass for energy or paper pulp as well as for seed oil extraction. Furthermore, wild and cultivated forms within Cynara genus are exploited for the extraction of biopharmaceuticals. Inulin, a natural fiber which was found to improve the balance of friendly bacteria in the intestine, can be extracted from roots, while leaves are very rich in antioxidant compounds. The latter: i.e. luteolin and cynarine, originating from the metabolism of phenylpropanoids, are able to protect bio-molecules (i.e. proteins, lipids and DNA) from oxidative damages caused by free radicals and play an hepato-protective as well as choleretic action, they might also contribute to the prevention of arteriosclerosis and other vascular disorders.

Up to know no information are available on genetic variation in natural population of wild cardoon and even less on chemical characteristics of leaf or root extracts with respect to different genotypes as well as soil and climatic conditions of growing areas. Furthermore, no research has been carried out on the genetic improvement of wild cardoon in order to maximize oil yields and biomass production.

We report on the genetic variation in eight natural populations of wild cardoon, of which five identified in Sicily and three in Sardinia, as measured by amplified fragment length polymorphism (AFLP) markers. 

Our results show that: (I) most of the genetic variation is present within populations, which is in keeping with the outcrossing breeding system of the species; (II) there is a remarkable tendency for plants of each population to cluster together, thus, despite the high ratio of within-to-between population genetic variance, the full AFLP banding pattern of each genotype is a relatively reliable fingerprint diagnostic of its parent population; (III) because of the confounding effect of habitat fragmentation, the geographical-genetic distance relationship is weak; (IV) the observed genetic distribution among populations provides insights for the identification of suitable strategies in order to sample genotypes representative of the natural genetic variation.

On the whole our results show that the high level of genetic diversity observed stress the need for the application of selection programmes in order to identify genotypes suitable for the extraction of biopharmaceuticals as well as the production of oil and biomass. The selected genotypes might then be tested in different environment in order to evaluate the effect of soil and climate on chemical composition of oil and leaf extracts, seed setting and biomass production. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.07

EMBRYO FORMATION FROM ANTHER CULTURES OF ANEMONE CORONARIA CULTIVARS

M. LAURA, G. SAFAVERDI, S. DOVERI, B. RUFFONI, A. ALLAVENA 

Istituto Sperimentale per la Floricoltura, Corso degli Inglesi 508, 18038 Sanremo (Imperia)

androgenesis, genetic resources, ornamental crops, plant breeding

Anemone coronaria is a popular ornamental species grown in southern Europe for the production of cut flowers. The species has in addition an attitude for the production of flowered pot plants. At present, cultivars are plant populations obtained by hybridisation of heterozygous parents. Recent success in micropropagation of parent plants allowed the production of population with similar plant composition through the years, but uniformity among plants is still lacking. Two strategies were developed to produce pure lines and then uniform F1 hybrid: selfing and androgenesis.

Selfing is a long way strategy considering that in cultivation Anemone is, a biannual plant and at least one-year time is required for each generation (seed to seed). In addition, some difficulties were observed in seed setting after one selfing generation.

In this study is report a protocol for embryo formation from anther culture of Anemone coronaria cultivars. Anthers of Cristina, Monalisa, Tetranemone and Wicabri, with pollen grain at uninucleate stage, were cultured aseptically in Petri dishes containing a double layer medium (Johansson and Eriksson; Physiol. Plant. 1977, 40:172-174) used for embryo formation from several wild Anemone species. The medium was based on the composition of Nitsch and Nitsch (Science, 1969, 163:85-87) salts plus activated charcoal (1%) with or without indol acetic acid (IAA, 0,1 mg/l). MS (Murashige and Skoog; Physiol.   Plant. 1962, 15:473-497) salts at half strength were also used in place of Nitsch and Nitsch medium. After twelve to fourteen weeks of culture, globular embryo-like structure developed from the inner anther tissues. Embryo-like structure rapidly developed into small rhizomes that finally germinate into plantlets. This development seems to be similar to plantlet ontogenesis from zigotic seeds. 

All the cultivars were able to regenerate plantlets on Nitsch and Nitsch medium, except White, Red and Fuchsia Wicabri. The highest frequency of globular embryo-like structures per 100 anthers occurred from cv. Cristina blue (11,3) on Nitsch and Nitsch medium without indol acetic acid. Very low or absence of regeneration was observed with cvs. White Cristina, Blue Wicabri, Purple Monalisa and White Tetranemone on medium without IAA and with cvs. Red Monalisa, Blue Monalisa, Purple Monalisa and Blue Tetranemone on medium with IAA. Embryo-like structure formation from anthers was also observed in MS salt at half strength. 
Further studies are in progress to verify if these plantlets take origin from pollen grain and then have haploid or double haploid chromosome number. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.08

USE OF RAPD MARKERS FOR THE GENETIC CHARACTERIZATION OF LIMONIUM WILD SPECIES

S. BRUNA*, G. BURCHI**, L. DE BENEDETTI*, A. MERCURI*, N. PECCHIONI***, C. AGRIMONTI****, T. SCHIVA*

*) Istituto Sperimentale per la Floricoltura, Corso degli Inglesi 508, 18038 Sanremo (IM), Italy
**) Istituto Sperimentale per la Floricoltura,Via dei Fiori 8, I-51012 Pescia (PT), Italy 
***) Istituto Sperimentale per la Cerealicoltura, Sezione di Fiorenzuola d'Arda-Via San Protaso 302, I-29017 Fiorenzuola d'Arda (PC), Italy
****) Dipartimento di Scienze Ambientali. Università di Parma, Italy

Limonium, RAPD markers, genetic similarity

The genus Limonium (fam. Plumbaginaceae) consists of about 300 species of mostly herbaceous perennials, some low shrubs, and annuals, natives chiefly of salt marshes, sea cliffs, semidesert and desert regions. Limonium is grown in many regions of the world for cut flower production for both fresh and dry-flower arrangements. 

RAPD analysis was used for the study of genetic relationships in Limonium. Thirteen wild species were tested with 10 primers. A total of 244 bands were scored and used for the analysis of genetic distances. The dendrogram obtained from cluster analysis showed an high similarity among three species that some authors report as synonymous and that in our phenotypic observations appeared very similar. In order to clarify the genetic relationships, further analyses were carried out on several genotypes belonging to the three species: L. caspia (12 accessions), L. otolepis (12 accessions), L. bellidifolium (8 accessions). In the dendrogram, obtained after screening RAPD bands, the genotypes belonging to each species appear to be grouped in three main clusters. AMOVA analysis showed that the percentage of variation within species (88,32%) is higher than variation between species (11,68%). Results are discussed.

The application of RAPD analysis in our case was thus an useful tool to clarify the taxonomy of the three Limonium species, in a plant genus that is notably of difficult interpretation

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.09

ANALYSES OF EXPRESSION OF A TYPE-II METALLOTHIONEIN-ENCODING GENE IN THE SEAGRASS POSIDONIA OCEANICA 

R. COZZA*, T. PANGARO*, P. MAESTRINI**, A. CAVALLINI**

*) Università degli Studi della Calabria, Dip.to di Ecologia Ponte P. Bucci, cubo 6B,  87030 Arcavacata di Rende (Cosenza)
**) Università degli Studi di Pisa, Dip.to di Biologia delle Piante Agrarie, Sezione di Genetica-Pisa

Posidonia oceanica, metallothioneins, in situ hybridization, heavy metals

The phanerogam Posidonia oceanica is endemic to the Mediterranean basin, growing as extended meadows in the infralittoral bottom and  playing a pivotal role for the maintenance of the coastal ecological equilibrium. It has been established that Posidonia is able to absorb and accumulate trace metals from marine environment thus controlling metal bioavailability in the marine ecosystem. All these findings  highlight  that this plant is a potential “biological indicator” of metal contamination. Concerning the response of marine angiosperms  to metals, so far studies have been mainly focused on their accumulation into plants tissues, whereas a few studies have been reported on the physiological and molecular processes triggered in these plants by metal exposure. As far as  Posidonia  is concerned, it has been shown that Cd++  exposure induces the synthesis of mRNAs encoding putative metallothioneins (Mts), small peptides, Cys-rich proteins that bind heavy metals.  In this work we have isolated a number of DNA sequences putatively encoding metallothioneins. Southern and sequences similarity analyses indicate that such DNA sequences belong to a multigene family of at least five members.

We have also determined,  by in situ tecnique, the expression domain of one type-II metallothionein gene in Posidonia plants in vivo. This gene shows a tissue specific expression being localized along the vascular bundle of the leaves; moreover the transcript show differences in expression in relation to the leaf axes, the leaf development and in the  shoot. Our results suggest that, at least some plant metallothioneins may have specialized functions confined to a few plant cell types.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.10

MOLECULAR EVOLUTION OF CATALASE GENE FAMILY IN THE GENUS POPULUS

S. CAPARRINI, P. DE BLASI, M. L. RACCHI 

Dip. Biotecnologie Agrarie, Università degli Studi di Firenze, p.le delle Cascine 24, 50144 Firenze

catalase, multigene family, phylogeny, Populus

Plant catalases are encoded by a small gene family. Studies performed comparing the exon-intron structures of known genomic sequences, indicate that intron positions are conserved and suggest the existence of an ancestral catalase gene containing seven introns. During evolution in plant species, the duplication of the primordial gene followed by the differential loss and gain of introns occurred to form three or four different isoenzyme genes. Unlike herbaceous plants, few molecular data on the catalase gene structure are available on woody plants. In this study we investigated the intron-exon structure of catalases of Populus species.

Because of its fast growth, ease for clonal propagation, strong heterosis upon interspecific hybridization, its small genome size (550 Mb) and its amenability for genetic transformation, Populus has become a model system for fundamental research on trees.

Using PCR approaches, three catalase genes have been identified and characterized in P. deltoides (section Aigeiros). Two catalase sequences contain seven introns in conserved positions, while one gene lacks the last four introns. Exon-intron structure of catalases was analysed for four species of the section Aigeiros (P. deltoides P. nigra, P. wislizenii, P. fremontii) and for three Leuce species (P. tremula, P. tomentosa, P. alba). 

The results indicate that the position of introns of each catalase is conserved, both in Aigeiros and Leuce sections. On the contrary, interesting differences were found in the size and sequence of introns of the same catalases, between the sections, and within each section, which will be used to examine the phylogenetic relationships among different species of the genus Populus.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.11

SEQUENCE DIVERSITY AND SNP MARKER DEVELOPMENT IN ALEPPO PINE (PINUS HALEPENSIS MILL)

G. G. VENDRAMIN*, M. ANZIDEI*, F. BAGNOLI**, C. PLOMION***, F. SEBASTIANI****, M. L. RACCHI**

*) IGV, CNR, Via Atto Vannucci 13, 50134 Firenze
**) DiBA, Università degli Studi di Firenze, Piazzale delle Cascine 24, 50144 Firenze
***) UMR BIOGECO, INRA, Pierroton, France
****) DISTAF, Università degli Studi di Firenze, Via San Bonaventura 13, 50145 Firenze

molecular markers, SNPs, population genetics, phylogenesis

Direct analysis of genetic variation at sequence level (Single Nucleotide Polymorphisms, SNPs) offers several advantages over other types of DNA marker systems. In particular, this approach allows gathering phylogenetic information through sequence variation analysis and drawing inferences on allele and population history that cannot be obtained with any of the other marker systems available. Information about the frequency and distribution of SNPs in forest tree species is still lacking. The objective of this work consists in a preliminary analysis of SNP distribution in Aleppo pine (Pinus halepensis) and in a first analysis of their usefulness in its population genetic analysis. Based on isozymes, nuclear and chloroplast microsatellite data it appears to carry high levels of variability, most of which (>85%) resides within populations. 

DNA extracted from seed endosperms that are haploid tissues were amplified and then sequenced. In this first phase attention was put on the CAD gene, which is involved in the lignin biosynthesis. Five populations sampled in different parts of the natural range of the species were analysed. Six endosperms for each population were used. The sequences were aligned using specific software, the single point mutations were identified, their frequencies estimated and the haplotypes were determined.

Based on preliminary data from about 1500 bp, the frequency of nucleotide changes appears to be high, with an average of one SNP every 150 bases overall. This frequency, which is much higher than that those observed in humans, appears to be similar that those observed in maize, Norway spruce and Maritime pine, which are commonly considered species with extremely high levels of variability. The estimated value of nucleotide variation appeared low. On the other hand the genetic differentiation among populations resulted higher than that estimated using neutral markers, suggesting that this gene is under selection.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.12

SEQUENCE CHARACTERISATION OF A VERY LARGE PLANT GENOME (NORWAY SPRUCE, PICEA ABIES (L.) KARST.)

A. ZUCCOLO, F. CATTONARO, I. JURMAN, M. VIZZI, M. MORGANTE

Dipartimento di Produzione Vegetale e Tecnologie Agrarie Università degli Studi di Udine, via delle Scienze 208, 33100 Udine

retrotransposon, ripetitive DNA, amplification, methylation

Genome expansion by amplification of repetitive sequences is a well described phenomenon. LTR-retrotransposons are the major contributors to the expansion in plants. Very large genomes (greater than 1*1010 bp per haploid genome) have however not yet been analysed at the sequence level to understand how the expansion was achieved, whether by amplification to very high copy number of a limited number of repeats or by amplification to lower copy numbers of a very large number of different repeats. The family Pinaceae in the Gymnosperms provides for an interesting case study because on one hand Gymnosperms are not yet well characterized at the genome level and on the other hand all species within the family have very large genomes.
 
In order to take a wide and comprehensive approach to the study of the structure of the Norway spruce (Picea abies (L.) Karst.) genome, seven different genomic libraries were constructed and used as source of clones to be sequenced. Three of them were screened to isolate by hybridisation a set of highly repetitive sequences: the 119 sequences obtained were characterised in silico and by Southern blot. Two other libraries were made using methylation sensitive enzymes to look at the hyper- and hypomethylated fractions respectively. Finally two libraries were made using randomly sheared (nebulised) genomic DNA to provide a random sample of the genome in one case and a random sample of its hypomethylated fraction in the other. Over 4000 sequences corresponding to more than 2 Mbp have been obtained from these last two libraries. 

Sequence analyses on all libraries reveal that: a) at least 70% of the genome is made of repetitive sequences; b) repetitive DNA is hypermethylated in Norway spruce; c) LTR retrotransposons are the major component of the repetitive fraction while other classes, although all present, are not abundant (LINEs, SINEs, tandem repeats); d) no single family of retrotransposons makes up a large fraction of the genome, while many families seem to have amplified to high levels. 

These results provide a different model for genome expansion in Gymnosperms from the one that seems to apply to cereals where single LTR families often make up 10% or more of their large genomes. We will discuss the results in light of the evolutionary history of Gymnosperms.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.13

DIVERSITY IN ALTERNATIVE PASTURE LEGUMES FROM ASINARA ISLAND FOR ANTI-NUTRITIONAL COMPOUNDS 

M. ODOARDI*, M. CONFALONIERI*, A. URSINO *, S. BULLITTA**

*) Istituto Sperimentale Colture Foraggere, Viale Piacenza 29, I-26900 Lodi
**) CNR-ISPAAM – Sezione Pascoli Mediterranei, Sassari, Via E. De Nicola, I-07100 Sassari

wild leguminous species, trypsin inhibitors, hemolytic saponins

The importance of pasturelands and forages, particularly for organic productions in extensive animal breeding systems, is increasing in Italy. As a consequence, a deeper knowledge is required about the phytotherapic as well as the antinutritive effects of natural compounds in pasture species grazed by animals. Such knowledge is still limited considering the richness of spontaneous plant species and of bioactive metabolites involved. These compounds can cause health problems and toxic effects to animals as in the case of antinutritionals, or can increase animal wellbeing  and productivity as in the case of bioactive phytotherapic compounds. 

In order to assess the potential of natural populations of leguminous species collected in Asinara island for their introduction into cropping in pasture improvement, a study was started to evaluate the presence and concentration of bioactive compounds in seeds and leaves of a number of natural species from a wider germplasm collection from Asinara. The chemical characterization of antinutritional compounds was mainly focused on: I) protease inhibitors, well represented among the seed storage proteins for their protective role against herbivore animals, and II) biologically active saponins, quite common in legume herbage, sometimes exerting anti-feedant and bloating effects on grazing animals. 

Seeds from eleven spontaneous species belonging to six legume genera have been considered for Trypsin Inhibitor (TI) content, while fresh leaves from the same accessions have been tested for biologically active saponins. Differences for TI content in seed and for hemolythic saponins in leaves have been detected among genera as well as among species. Different future uses of such wild species can be so suggested in pasture improvement, soil protection and multiple uses in Mediterranean environments, also in accordance with the rules of organic animal productions, based on providing maximum animal wellbeing and human health and on reducing environmental impact. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.14

VECTOR FAMILY FOR EFFICIENT TARGETING OF FOREIGN GENES TO TOMATO CHLOROPLAST DNA

F. VANTINI*, C. GOVONI*, L. CATTIVELLI**, M. GASTALDELLI*, G. TACCONI**, R. BASSI*** 

*) Laboratorio di Fotosintesi, Dipartimento Scientifico Tecnologico, Università di Verona
**) Istituto Sperimentale per la Cerealicoltura, Fiorenzuola d’Arda
***) Universitè Aix-Marsille II, LGPB, Facultè des Sciences de Luminy

chloroplast, vector, transformation, tomato

Genetic information in plants is localised in three subcellular organelles: nucleus, mitochondria and plastids. For many years, biotechnological strategies were focalised on nuclear genome transformation. More recently, plastid genome (cpDNA) has become amenable to genetic engineering. Indeed, high number of chloroplasts per cell and their elevated ploidy coupled with the absence of gene silencing and position effect, get to reach unexpected high expression level of transgene. Moreover, prokaryotic-like genes organisation allows multiple genes expression with single transformation event, and maternal inheritance in most agronomical species makes plastid transformation technology environmental safe.  

The production of recombinant proteins in plants has many potential advantages for generating biopharmaceuticals relevant to clinical medicine. One attractive advantage offered by the high level of foreign protein expression concerns the employment of plant edible organs in direct oral administration of active principles, e.g. edible vaccines. Therefore, we chose to induce plastid transgene expression in tomato (Lycopersicon esculentum) berries, where chloroplasts accumulate in green fruit and evolve to cromoplast during ripening. 

We developed a family of tomato chloroplast integration vectors (pZAI). Expression cassettes contain multiple cloning site (MCS) at 5’ position and, downstream, two markers conferring resistance to spectinomycin (aadA) and betaine aldehyde (bet B). The operon is expressed from the 5’-(P) and 3’-(T) regulatory regions of plastidic genes: Prrn, the strong promoter of rRNA (rrn) plastidic operon and TpsbA, the 3’ region of psbA. Every expression cassette is surrounded by two flanking regions (left, FRL and right, FRR) that drive the insertion via cpDNA homologue recombination. In order to design specific FR, we sequenced a 5 Kb region of tomato cpDNA inverted repeats and we cloned FR in pZAI vectors (respectively 0.8 Kb and 1 Kb) that localise the insertion between trnI and trnA genes.

Plasmids were synthesised using Fusion PCR technique that allows to join all the cassette’s elements saving time in cloning. Finally, markers were tested in E. coli, because of Prrn works as well in plastids as in prokaryotes. Bacteria were plated on LB + Spect200 medium and BADH activity was colorimetrically measured with NAD+ reduction assay. In both experiments transformed E. coli proved to express active marker proteins.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.15

HIGH OLEIC SUNFLOWER VARIETIES FOR BIODIESEL: A NEW PERSPECTIVE FOR SUNFLOWER CROP

M. BALDINI, M. VISCHI, N. DI BERNARDO, M. TURI, GP. VANNOZZI, A.M. OLIVIERI

Dipartimento di Produzione Vegetale e Tecnologia Agrarie, Università di Udine, via delle Scienze, 208, 33100, Udine

sunflower, biodiesel, high oleic, Sclerotinia sclerotiorum

The interest in many oil seed species as diesel fuel substitutes (Biodiesel) is increasing and various oil containing crops are grown for this purpose. The process of obtaining biodisel is very easy, involving a reaction of a vegetable oil with ethanol or methanol, in the presence of a catalyst to produce an ester (biodiesel) and glycerin (a valuable coproduct). Biodiesel can substitute for petroleum diesel fuel in most diesel engines with none or only minor modifications. It is biodegradable, renewable and nontoxic, with a reduced level of pollutants and safer than the diesel with a higher melting point (118 and 54 °C, respectively). Moreover no appreciable differences between biodiesel and diesel in engine durability or in carbon deposit have been demonstrated. Biodiesl production and commercial use in EU has expanded with some exemptions for biodiesl from excise taxes in many EU countries (Italy included) with a plan to develop a 5% market share for biodiesel by the year 2005. The commercial success of such fuels depends on their oil physical and chemical characteristics. Sunflower is one of the leading oilseed crops for the oil production for human consumption, but it has also been considered as an important crop for biodiesel production, particularly in southern European countries. In particular high oleic sunflower oil, because of low suscepibilty to oxidative changes during refining, storage and frying has not only a great value for human nutrition but it could be also an excellent renewable raw materials for industrial utilization, as lubricants for engine and particularly as biofuel (biodiesel). In fact the oil fatty acid profile with the degree of saturation influence two of the most important parameters for biodiesel: Cold-Filter Plugging Point (CFPP) related to the low temperature behavior and the iodine number, affecting the tendency of oxidation and polymerization of the oil. Therefore, new sunflower genotypes with a high oleic acid content in combination with reduced levels of linolenic acid are highly desirable. Since some years Udine University is involved in researches with the aim of obtaining new high oleic sunflower varieties, using the mutant gene for high oleic content present in Pervenet variety. Up to present two new varieties, named Friuli and Carnia, were registred in the Italian Register. These varieties were tested for agronomic traits and oil quality in 4 locations during summer 2002 and the performances were very satisfactory. New varieties are also under field trials for inscription in the Register. In the mean time we are carring out a molecular analysis to find out SSR markers associated to Slerotinia sclerotiorum resistance and to develop a MAS approach to introduce resistance in high oleic sunflower lines, as S. sclerotiorum is one of the most dangerous and complicated crop pathogens to control. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.16

IN PLANTA EXPRESSION OF A FUNGAL LACCASE GENE USEFUL FOR PHYTOREMEDIATION OF PAHs CONTAMINATED SOILS

GALANTE C., ESPOSITO S., STILE M.R., MONTI L., FILIPPONE E.

Department of Soil, Plant and Environmental Sciences, School of Biotechnology, University of Naples “Federico II”, Via Università 100, 80055 Portici (Italy)
cgalante@unina.it, filippon@unina.it

laccase, poxC, phytoremediaton, PAHs, transgenic plants

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and relocated in the environment as a result of the incomplete combustion of organic matter. Many PAHs are highly toxic, mutagenic and/or carcinogenic to microorganism as well as to higher organisms, including humans. Plants have been claimed to have the capability to produce both degradation enzyme in root exudates and a better environment for the growth of useful microbia in the rhizosphere, allowing them to exert their degradation potentiality. To enhance plants PAHs degradation ability, our research approach was to express in planta fungal phenol-oxidases (laccases), which, released by means of exudates roots, degrade condensed PAHs into less toxic compounds. The poxC laccase-encoding gene1, from the white-rot-fungus Pleurotus ostreatus, was used in our experiment. In silico analysis of POXC peptide sequence predicted the extracellular localization of this enzyme in plant. The cDNA of poxC gene was inserted into the pKYLX binary vector, under the CaMV35S2 constitutive promoter, then the vector was transferred into Agrobacterium tumefaciens LBA4404 strain and used for agro-infiltration and for stable genetic transformation experiments of Nicotiana tabacum cv Samsun NN. The western analysis on total protein extract from agro-infiltrated leaves confirmed the presence of the transgenic enzyme. The regenerated shoots, selected in presence of kanamycine, were analysed by PCR, using specific primers for the poxC gene and for the A. tumefaciens vir genes region: all putative transgenic shoots were only positive to the amplification of poxC gene. The RT-PCR analysis confirmed the transcript presence in transgenic plants and pointed out different levels of specific transcript. Transgenic plants, growing in vitro conditions, were phenotypically different from the control untransformed ones. In particular, they were smaller than controls and they showed leaves with red-brown patches. However, roots growth and morphology were comparable with control plants. The observed differences in growth and in morphology between the organs of the transgenic plants in comparison with the untransformed ones should be related to the interference of the over-produced laccase on tobacco lignin biosynthesis. Other experiments are in course to ascertain the enzymatic activity of root exudates and to drive poxC gene expression into plant roots only.

Acknowledgements: we thank G. Sannia and P. Giardina (Dept. of Organic Chemistry and Biochemistry, University of Naples “Federico II”, Italy) for providing us the P. ostreatus poxC gene.

References
1 Giardina et al. (1999) Biochem. J. 341: 655-663

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.17

Genetic characterization of native germplasm of Salix alba L. from Brenta river for multiple use land reclamation

S. Meneghetti*, G. Barcaccia*, P. Paiero**, M. Lucchin*

*) Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Università di Padova – Agripolis, via Romea 16, 35020 Legnaro, Padova, Italy
margherita.lucchin@unipd.it
**) Dipartimento Territorio e Sistemi Agroforestali (TESAF), Università di Padova.

Salix alba, AFLP, genetic variability, linkage disequilibrium
 
Environmental exploitation has profoundly affected the quality of the landscape and its natural features, disturbing the ecological balance and destroying important habitats. However, a stronger concern is finally arising for a better management of the landscape; tree woodlands, natural sites are more and more considered for their natural and multiple use values.

Recently, many projects have been developed to rebuild a woody vegetation cover on disturbed or marginal sites, with both an environmental and a multiple use purpose. A careful selection of species and populations to use, on the basis of an accurate ecological and genetic evaluation, is necessary to guarantee that good results are obtained.

Our research deals with the evaluation of genetic variability in natural populations of Salix alba L., a colonising tree species, from the middle course of Brenta river (North-East Italy) and the study of relationships between genetic and morphological variation in order to detect some chromosomal regions affecting quantitative traits. A comparative characterization of three natural populations of S. alba from Fontaniva (VI), Carturo (PD) and Piazzola sul Brenta (PD) was carried out using AFLP molecular markers. 

Selected primer combinations generated a total of 276 amplified products, 178 of which showed to be polymorphic (64.5 %). The Shannon’s diversity information index over populations and AFLP loci was I = 0.251.  The mean Dice’s genetic similarity coefficients within and between populations were similar and equal respectively to 0.887 and 0.885; the same was true for the mean genetic distances that were respectively 0.120 and 0.122. These results were in agreement with the amount and the partition of genetic diversity. The total Nei's genetic diversity was HT = 0.159, while that calculated for single populations was, on average, HS = 0.151. The extent of differentiation between populations was GST = 0.050 showing that as much as 95% of the total genetic variability observed can be attributed to within-population differences and only 5% is due to differences among the three populations, as confirmed by the high estimated value of gene flow (Nm = 9.50).

Relationships between molecular polymorphisms and phenotypic variation in traits were assessed by the detection of linkage disequilibrium between selected AFLP loci. Significant (P<0.05) pair-wise linkage disequilibria were found for all the morphological traits but stem diameter. The number of linkage disequilibria was ranging from 1 of both stem straightness and stem inclination to 4 of stem taper. On the whole 12 significant pair-wise linkage disequilibria were found. These results, together with the R2 coefficients of multiple regression analyses ranging from 0.22 to 0.46, allowed to identify some chromosomal regions where QTLs supporting large phenotypic effects are located. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.18

TRANSIENT EXPRESSION OF CLASSICAL SWINE FEVER VIRUS A1-EPITOPE IN NICOTIANA BENTHAMIANA PLANTS INFECTED WITH RECOMBINANT POTATO VIRUS X

G. MARCONI*, A. PORCEDDU**, P. BARONE*, E. ALBERTINI*, D. RUTILI***, F. VERONESI*

*) Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Università degli Studi di Perugia, Borgo XX Giugno 74, 06121 Perugia
**) IRMGPF-CNR, Via Madonna Alta 130, 06128 Perugia
***) Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Via Salvemini 1, 06121 Perugia 

PVX, transient expression, CSFV

Plant viruses have been used as vector for peptide and proteins production. The high expression levels and the possibility of exposing on the coat surface foreign peptide allow their use as vaccines.The vector used in the present study is Potato Virus X (PVX), a filamentous plant-virus infecting many members of the Solanacee family including potato, tomato and pepper. 

The Classical Swine Fever Virus (CSFV) is an economically important infectious disease occurring in pigs causing direct and indirect losses. In Europe it is estimated that more than 2 milion of pigs die per year. The etiological agent of CSFV is a virus of the genus Pestivirus (Flaviviridae family). Previous studies have shown that the glicoprotein E2 is highly immunogenic with four antigenics domains A to D. The domain A is divided in subdomains A1, A2 and A3. Subdomains A1 and A2 are conserved in more than 90 CSFV strains, but only subdomain A1 is neutralizing.. 

The PVX cDNA was modified through the fusion to the N-terminal portion of CP of a 16 amino acid sequence, called FMDV 2A and subdomain A1 the CSFV to produce the 35SPVXA12ACP construct. The FMDV 2A sequence allow to obtain a proteolityc cleavage with an efficiency of about 50-70%. 

Nicotiana benthamiana plants were infected with virus carring plasmid 35SPVXA12ACP or wild-type (WT) PVX by abrading the surface of two leaves with carborundum and inoculating each leaf with 25 g of plasmid DNA. Upon sistemic infection (10 to 15 days after inoculation) the correct expression of construct was verified through RT-PCR. The recombinant protein was analized through Maldi-Tof method. Sera from rabbit immunized with 35SPVXA12ACP showed IgG specific for E2 peptide, while no reactivity was found in the sera of control animals.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.19

RATIONAL DESIGN AND PRODUCTION OF INTRACELLULAR ANTIBODIES WITH DESIRED SPECIFICITY

M. DONINI*, V. MOREA**, A. DESIDERIO*, D. PASHKOULOV*, M. E. VILLANI*, A. TRAMONTANO***, E. BENVENUTO*

*) ENEA, UTS Biotecnologie, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, 00100 Roma, Italy 
**) CNR Center of Studies of Molecular Biology, Piazzale Aldo Moro 5, 00185 Rome, Italy
***) Department of Biochemical Sciences 'A. Rossi-Fanelli', University 'La Sapienza', Piazzale Aldo Moro 5, 00185 Rome, Italy

ScFv, intrabodies, CDR grafting, immunotherapy

The ability of antibodies to recognise a virtually infinite number of molecules with high affinity and specificity makes them invaluable tools for both experimental and therapeutic applications. In particular recombinant mammalian antibodies and their derivatives, have been expressed in plants to provide the so called “plantibody-based resistance” (Schillberg et al., 2001, Transgenic Res. 10, 1-12) against a number of different pathogens, including viruses, bacteria, fungi and invertebrates. 

The first antibody-mediated virus protection in plants was reported by Tavladoraki et al. (Tavladoraki et al., 1993 , Nature 366, 469-472). A single chain Fv (scFv(F8)) fragment (specific for artichoke mottled crinkle virus, AMCV) was constructed from a monoclonal antibody and expressed in bacteria showing to retain  antigen-binding specificity. The recombinant antibody was then expressed in the plant cytoplasm and both transgenic plants and protoplasts proved to be protected from virus infection. 

We wanted to investigate whether reshaped antibodies with new specificity can be rationally designed that retain cytoplasmic solubility and functionality of  the stable scFv(F8) molecule. 

We present here the design and production of a 'reshaped' antibody obtained by grafting the antigen binding site of the anti-lysozyme D1.3 antibody into the framework regions of the scFv(F8) fragment. The original scFv(F8) neutralises its cognate antigen, the plant virus AMCV, in the plant cytoplasm and it binds the AMCV coat protein in the cytoplasm of bacterial and eukaryotic cells (Tavladoraki et al., 1999, Eur. J. Biochem., 262, 617-624.). These are very peculiar features, in that the large majority of antibodies and antibody fragments are neither stable or functional in the highly reducing cytoplasmic environment. At the same time, these features are very desirable, in view of the very large number of potential applications of intracellular antibodies in research studies and human therapy (Desiderio, A., et al., 2001, J. Mol. Biol. 310, 603-615).

The reshaped scFv(F8)-D1.3 molecule that we designed was found to be able to bind lysozyme with affinity comparable to that of the antigen binding site donor D1.3 both in the oxidized form, obtained by periplasmic extraction, and in the reduced state, obtained by cytoplasmic extraction under reducing conditions. This demonstrates that the framework regions of the scFv(F8) are able by themselves to produce the cytoplasmic stability of the original scFv(F8). These regions can therefore be used as a scaffold to engineer antibody fragments that are stable and functional in the cytoplasm and endowed, in principle, with specificity against any particular plant pathogen. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.20

SELECTION OF SUNFLOWER CELL CULTURES WITH IMPROVED ALPHA-TOCOPHEROL BIOSYNTHESIS

C. FACHECHI, R. GALA, G. ZACHEO, S. CARETTO

Istituto di Scienze delle Produzioni Alimentari, Sezione di Lecce, CNR, Via Prov.le Lecce-Monteroni 73100 Lecce, Italy

vitamin E, in vitro production, tocopherol, chlorophyll, sunflower

Tocopherols (vitamin E), one type of antioxidant phytochemicals in plants, are known to prevent degenerative disease processes in humans. One particular form, lpha-tocopherol, is the most biologically active component of vitamin E and is synthesised in its effective form only by photosynthetic organisms. Vegetable oils and, to a lesser extent, vegetables are the main dietary sources of lpha-tocopherol. 

In plants, lpha-tocopherol acts mainly as a highly efficient recyclable chain reaction terminator for the removal of the polyunsaturated fatty acid free radicals, which are generated during lipid peroxidation. The biosynthesis of lpha-tocopherol occurs in chloroplasts of higher plants where it is especially localised.

Using sunflower cell cultures, growing in heterotrophic conditions, an in vitro system for the biotechnological production of natural lpha-tocopherol was established.

Among the obtained sunflower cell cultures, two suspension cell lines having different levels of alpha-tocopherol were identified: one cell line (HT - High Tocopherol) had an almost 3-fold higher tocopherol level than the other (LT – Low Tocopherol). The chlorophyll content was analysed in both lines  resulting significantly increased in HT cells compared to LT cells. Cytological evidences revealed the presence of a higher number of chloroplasts in HT cells than in LT. The ability to grow in the presence of reduced sucrose concentration (mixotrophic conditions) was tested for both cell lines. While LT cells could not survive with a reduced exogenous carbon source, HT cells not only could cope with the sucrose-stress but also showed an improved ability to biosynthesise both chlorophyll and lpha-tocopherol. The cell growth time course of HT growing in mixotrophic conditions is in progress and will help discussing the efficiency of this system for the biotecnhological production of lpha-tocopherol.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.21

TOBACCO PLANTS AS BIOFACTORIES FOR THE PRODUCTION OF AN ANTI-HIV VACCINE

E. FERRAIOLO*, L. BUONAGURO**, F.M. BUONAGURO**, M.L. TORNESELLO**, L. MONTI*, N. SCOTTI*, S. GRILLO*, T. CARDI*

*) CNR-IGV, Institute of Plant Genetics, Res. Div. Portici
cardi@unina.it
**) Viral Oncology, Ist. Naz. Tumori “Fond. G. Pascale”, Napoli

tobacco, biopharmaceuticals, biofactories, vaccines, transgenic plants

Tobacco, an important crop in various Italian Regions, is an attractive plant for the production of Plant Made Pharmaceuticals, since it is a no-food crop and thus there is no risk of cross contamination of the food chain. It has a large biomass and very good response to various biotechnological approaches. Further, its cultivation for the traditional use as drug, subsidized by European Union, does no longer remunerate farmers and it is under close scrutiny.

In Western Countries, the HIV, the well known agent of the “Acquired ImmunoDeficiency Syndrome” (AIDS), is presently controlled by anti-retroviral therapeutic strategies (HAART). Nevertheless, the definitive control of the disease is still difficult due to the high frequency of “escape mutants” and the unavailability of the HAART therapy in developing countries. An alternative strategy relies on the development of preventive anti-HIV vaccines. A promising approach is based on the property of the capsid protein Gag to auto-assemble and form Virus-Like Particles (VLPs) displaying several viral epitopes on the surface. Encouraging results have been already obtained by producing VLPs expressing gag-pol-nef and env genes in the baculovirus/insect cell expression system (Buonaguro L. et al. 2001 Antiviral Res. 49, 35-47; Buonaguro L. et al. 2002 Antiviral Res. 54, 189-201).

In order to test the feasibility to produce a similar range of viral antigens in plant cells, we recently cloned a modified gag-pol-nef gene construct in an Agrobacterium-based plant expression vector, and the env gene in a plant vector suitable for biolistic transformation. In the former case selection was based on a cointegrated nptII gene conferring kanamycin resistance, whereas in the latter it depended on a hpt gene, determining hygromycin resistance, on a separate plasmid. Expression of both transgenes was driven by the constitutive 35S CaMV promoter.

We obtained transgenic plants with both constructs and approaches. In experiments with Agrobacterium tumefaciens, from 30 co-cultivated explants we obtained 23 independent KanR shoots that were also positive to PCR analysis; that is equivalent to a transformation frequency of 0.8 transgenic shoots per explant. In co-transformation experiments using the biolistic approach, we bombarded explants with different molar ratios of two plamids, carrying the hpt and the env genes, respectively. Transformation frequencies ranged from 2.4 to 9.8 independent hpt+ shoots per bombarded explant. Co-transformation frequencies varied from 33 to 57%. Best results, both for the  integration of the marker gene and for the co-transformation efficiency, were obtained with a 1:8 (hpt:env) ratio between the two plasmids.

Further molecular characterization (RT-PCR) of transgenic plants indicated that both gag and env genes were transcribed in the tobacco cells, albeit at relatively low levels. Plants with higher production of the transcript could be selected. Analysis of protein expression is under way.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.22

STRATEGIES FOR THE IDENTIFICATION OF BIOACTIVE MOLECOLES IN PASSIFLORA SSP.

F. GUZZO*, S. CEOLDO*, F. ANDREETTA*, A.M. MARCONI**, M. LEVI*

*) Università di Verona, Dipartimento Scientifico e Tecnologico, Strada le Grazie 15, Cà Vignal 1, 37134 Verona
**) Laboratori Agro-Alimentari Consorzio ZAI, Via Sommacampagna, Verona

Passiflora ssp, bio-guided fractionation, antimicobial activity

The genus Passiflora comprises several hundred species, grouped in 21 subgenera. Only some species of the genus (P. edulis, P. quadrangularis, P. ligularis) are widely studied for their economic importance and are chiefly cultivated for production of fruit juice. P. incarnata is well known for its sedative properties and several other species are of ethnobotanical importance (see The Phytochemical and Ethnobotanical Databases, at http://www.ars-grin.gov/cgi-bin/duke/ethnobot.pl). Most of the species are almost completely unstudied. 

Unfortunately, in temperate countries, plants of only few species of the Passiflora genus are available and are commonly grown as garden or pot plants. For most of the Passiflora species, only the dehydrated seeds can be readily purchased. However, dehydrated seeds of many Passiflora species may require many months, and up to two years to germinate (Vanderplank, Passiflora Society International Meeting, Rome, 15-16 september 2001). In order to produce an in vitro culture collection of Passiflora species together with a green house collection of plants as a source of starting material, we set up a zygotic embryo culture technique. Of the 61 species available, we obtained stable undifferentiated calli from twenty eight species, fourteen of which also regenerated plants; for five of these fourteen species (P. foetida, P. palmeri, P. coriacea, P. tenuifila, P. apetala) regenerated plants material was enough for chemicals extraction and bioactivity determination. One more specie (P. nitida) did not regenerate but produced one plant through in vitro embryo germination, and this plant was .screened as well for the biological activity.

A biological assay was developed for screening of potential antibacterial compounds, based on growth inhibition of wild type E. coli strains. In this assay the growth inhibition is determined through the spectrophotometric determination of broth turbidity at 600nm. As initial screening, crude methanolic extracts from the 6 Passiflora species indicated above were tested, and two species (P. palmeri and P. nitida) showed to have antibacterial activity. The assay was also used for bio-assay guided fractionation of the extracts. The crude extract from P. nitida was fractionated by Solid Phase Extraction and HPLC, and only one active fraction was identified in both cases.

Cytometric determination of live and dead bacteria showed that the extracts caused cell death very rapidly.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.23

BIOACTIVE COMPOUNDS IN CITRUS GENOTYPES: PRESENCE AND BIOLOGICAL FUNCTION. PRELIMINAR RESULTS

S. FATTA DEL BOSCO*, L. CAMARDA**, V. DI STEFANO**, D. SCHILLACI**

*) Istituto di Genetica Vegetale, sezione di Palermo, C.N.R.
**) Dipartimento di Chimica e Tecnologie Farmaceutiche, Università di Palermo

genotype, phytochemicals, flavonoid

There is currently much interest in phytochemicals as food bioactive components. Numerous epidemiological surveys showed a direct relationship between fruit, vegetables and cereals intake and protection against cardiovascular disease and cancer. Among the different mechanisms that contribute to the protective properties, the anti-oxidant (radical-scavenging) activity is considered a significant and crucial factor. 

Recent work is emphasizing the role of flavonoids as important anti-oxidant components. Citrus fruit are particularly rich in flavonoids compounds, mainly stored in the vacoules as flavanone glycosides; their amount and characteristics are highly genotype-specific.

Orange and grapefruit juices (as well as the fruits themselves) are highly consumed  and are potentially major contributors to the total dietary flavonoids. 

We have analyzed the following species and varietes: Sweet orange (Citrus sinensis L. Osb.) cvs. Washington Navel and Vaniglia, Mandarin (C. reticulata Blanco) cv. Avana, Grapefruit (C. paradisi L.) cv. Ruby Red Star, Clementine (C. clementina Hort. Ex Tan.) cv. Nules, Bitter orange (C aurantium myrtifolia).
 
Centrifuged fresh juices, added with an internal standard, were analyzed by HPLC with UV-Vis detector. By comparing retention times with commercial standards, five flavanone glycosides were identified and quantitated.

Differences have been observed in terms of flavanone glycoside content in the genotypes analyzed.

The fruit juices were tested for their in vitro antiproliferative activity against K562 (human chronic myelogenous leukemia), L1210 (murine leukemia) and MCF-7 (human breast adenocarcinoma) cell lines. The antiproliferative effects were estimated in terms of growth inhibition percentage at a screening concentration of 10%v/v.

Preliminary results are interesting, in fact some Citrus varietes showed a value of growth inhibition percentage of 100% against leukemic cell lines and about 80% against MCF-7 cell lines.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.24

PLANT-DERIVED SEQUENCES AND PLANT VIRAL GENES FUSED WITH A MODIFIED HPV16 E7 GENE FOR THE DEVELOPMENT OF NEW DNA THERAPEUTIC VACCINES AGAINST HPV-RELATED TUMORS 

S. MASSA*, P. SIMEONE**, E. ILLIANO*, E. BENVENUTO*, A. VENUTI**, R. FRANCONI*

*) ENEA, BIOTEC GEN, CR Casaccia, Via Anguillarese 301, 00060 Rome, Italy
**) Istituti Fisioterapici Ospitalieri (IFO) - Istituto Regina Elena per lo Studio e la Cura dei Tumori, Via delle Messi d'Oro 156, 00158 Rome, Italy

Human Papilloma Virus 16, E7 oncoprotein, Potato Virus X, coat protein  

The high-risk types of Human Papillomavirus (HPV16, 18) are currently accepted as the primary etiologic agents of cervical cancer. Being the HPV16 E7 oncoprotein constitutively expressed in these tumor cells and responsible for the onset and maintenance of the transformed state, it represents a promising target for the development of therapeutic vaccines against HPV-associated malignancies.

DNA vaccination is an attractive approach for HPV immunotherapy and, in pre-clinical studies, it has shown to elicit long-term humoral and cellular immunity. Clinical trials are also in progress. 

Besides their exploitation as biofactories, plants and plant viruses can offer new opportunities to increase the efficacy of DNA vaccines by providing sequences that are primary antigen in humans. 

In this perspective, we firstly introduced three point mutations into the retinoblastoma (pRb)-binding site of HPV16 E7 oncogene to abolish its transformation potential (“E7GGG” gene). Then we made N- and C-terminal gene fusion of the E7GGG gene with, respectively, the Potato Virus X coat protein (PVX-CP) and the signal peptide coding sequence of the polygalacturonase-inhibiting protein (PGIP) from bean. 

The former gene fusion should exhibit high immunogenicity. In fact, it has already been shown that such an approach with other tumor associated antigens, leads to highly aggregated, self-assembling structures able to increase the CD4+ immune response1. The E7GGG sequence from HPV16 was directly assembled with the PVX-CP or fused in frame to the fourth codon of PVX-CP via the aminoacid linker ”Gly-Pro-Gly-Pro”.

We already reported the enhanced expression level of a secretory HPV16 E7 protein (PGIP-E7) in N. benthamiana plants. Fusion to the PGIP sequence in a DNA vaccine, should improve E7 protein expression level and “visibility” by the immune system.  

The synthetic genes were cloned into a mammalian expression vector. The above mentioned DNA constructs are currently being tested in order to assess whether they induce humoral and/or cellular immune responses and protection from cancer in vaccinated mice challenged with a tumorigenic dose of E7-expressing singeneic tumor cells. Data on biological effects will be presented.

1. Savelyeva N. et al. (2001) 'Plant viral genes in DNA idiotypic vaccines activate linked CD4+ T-cell mediated immunity against B-cell malignancies'. Nature Biotechnology 19: 760-764.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.25

POTATO VIRUS X CHIMERIC PARTICLES FOR VACCINE DEVELOPMENT

C. LICO*, C. MARUSIC**, F. CAPUANO*, P. RIZZA***, F. BELARDELLI***, E. BENVENUTO*, I. CAPONE***, S. BASCHIERI*

*) ENEA, UTS Biotecnologie, Protezione della Salute e degli Ecosistemi, Sezione di Genetica e Genomica Vegetale, C.R. Casaccia, Rome
**) ENEA, UTS Biotecnologie, Protezione della Salute e degli Ecosistemi, Sezione di Genetica e Genomica Vegetale, C.R. Triasaia, Rotondella (Mt), Italy
***) Istituto Superiore di Sanità, Laboratorio di Virologia, Rome, Italy

PVX, chimeric virus particle, vaccine, HIV-1 epitope

In the last decade the use of genetically modified plant viruses has been explored as efficient tool to express in plant tissues heterologous proteins of biomedical interest.  A particular application of this technology is finalized to the production of innovative vaccines by generating chimeric virus particles (CVPs) expressing on their coat protein (CP) pathogen-derived epitopes (i.e. short aminoacid stretches known to be responsible of the activation of the immune response against a pathogen). In this case the viruses are not only a plant expression system but also fundamental vaccine components working as delivery  tools. 

We have previously demonstrated that the mucosal delivery of purified Potato Virus X (PVX) chimeric particles expressing the 2F5e epitope of HIV-1 gp160 protein, is able to induce strong IgA and IgG antibody responses in mouse models, without the need of adjuvants. 

We are presently trying to improve this vaccine delivery system by modifying  the CP of PVX to display longer immunostimulating epitopes.
   
Two modified viral expression vectors, derived from pPVX201, have been constructed to insert, as CP-fusion, HIV-1-derived epitopes of different lengths. The modified vector pPVXCC has been used to display epitopes able to induce neutralizing antibody-responses (8, 14 and 23 aminoacids) while the modified vector pPVXSMA has been used to display epitopes known to induce T-cell mediated immune responses (9 and 10 aminoacids). 

The viral vectors (infectious transcripts) have been used to infect N. benthamiana plants.  Ten days after infections RNA have been extracted  from systemically infected leaves and reverse transcribed. Sequence analysis have been performed on PCR fragments amplified with PVX-specific primers. 

Both pPVXCC and pPVXSMA are able to systemically  infect plants, producing symptoms that are identical to those induced by pPVX201. Moreover, they both replicate stably in infected tissues, as assessed by repeated infection cycles with infected plant extracts.   

The pPVXCC derived vectors carrying the sequence coding for 8 or 14 aa long epitopes (as opposite to the vector carrying the sequence coding for the 23 aa long epitope, which seems to be unable to start plant infection) generates CVPs able to systemically  infect plants, without evident symptoms. Nonetheless both CVPs display correctly the exogenous sequences.

In the case of the pPVXSMA derived vectors, only the CVPs displaying the 9 aa epitope ends up in systemic infections, with symptoms undistinguishable from pPVXSMA and pPVX201, retaining the exogenous sequence.

From these results we can conclude that both modified vectors can be considered as interesting alternatives to pPVX201 for the production of CVPs displaying hexogenous sequences that are in the case of pPVXCC 14 aminoacid residues long and in the case of pPVXSMA 9 aminoacid residues long. However it is important to note that successful epitope display on hybrid CP is affected not only by the length of the sequence but also by its “quality” in terms of aminoacid composition.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.26

BOOSTING HUMAN GAD EXPRESSION IN TRANSGENIC PLANTS

L. AVESANI*, A. FALORNI**, E. DALLA POZZA*, G.B. TORNIELLI*, F. MORANDINI*, L. BORTESI*, M. PEZZOTTI*

*) Dip.Scientifico e Tecnologico, Università degli Studi di Verona
**) Dip. di Medicina Interna, Sezione di Medicina Interna e Scienze Endocrine e Metaboliche, Università degli Studi di Perugia

autoimmunity, GAD65, oral tolerance, transgenic plants

The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. Successively, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by the mediating expression through the potato virus X (PVX) vector. By substituting the NH2-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD67/GAD65 molecule was expressed via-Agrobacterium in transgenic tobacco plants. Immunolocalisation analysis confirms cytosolic and a radio-immunoassay analysis demonstrates that expression levels of hGAD65 were increased five times compared with that obtained in the previous stable transformation (0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65). Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. 

In the present study we report new strategies aimed to boost hGAD65 expression in tobacco plants in order to plan oral tolerance studies on animal models. To increase stable expression of hGAD65 in plants Agrobacterium-mediated gene transfer expression vectors were developed. By using such vectors we tested the hypothesis that the expression levels of hGAD65 could be enhanced by targeting the human protein to the ER and by using an enzymatic inactive form of the recombinant protein.

Moreover, to express hGAD65 in edible plant organs, transgenic tomato plants were also generated; therefore we constructed an Agro-based vector in which hGAD65 expression was driven by a tomato fruit-specific promoter.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.27

EXPRESSION OF THE VITREOSCILLA HEMOGLOBIN (VHb)-ENCODING GENE IN TRANSGENIC WHITE POPLAR: BIOCHEMICAL CHARACTERIZATION AND EFFECTS ON PLANT GROWTH RATE AND BIOMASS PRODUCTION

S. ZELASCO*, S. REGGI**, S. BISOFFI*, C. BONGIORNI***, P. CALLIGARI*, A. BALESTRAZZI****, C. FOGHER***, M. CONFALONIERI*****

*) Istituto di Sperimentazione per la Pioppicoltura, MiPAF, Casale Monferrato (AL)
**) Plantechno Srl, Casalmaggiore (CR)
***) Istituto di Genetica Vegetale - UCSC, Piacenza
****) Dipartimento Genetica e Microbiologia, Pavia
*****) Istituto Sperimentale per le Colture Foraggere - MiPAF, Lodi

biomass production, hemoglobin, genetic engineering, Populus, Vitreoscilla

Increasing growth rate, biomass production and wood quality of trees represent a main goal in most breeding programs worldwide. Constitutive over-expression of genes encoding proteins involved in the aerobic cellular metabolism is one of the strategies proposed to modify and improve these important agronomic traits. We describe a first attempt to increase vegetative growth and yield production in transgenic poplar by expressing a Vitreoscilla (VHb) hemoglobin-like protein. A chimeric construct consisting of the cauliflower mosaic virus 35S promoter and the coding region of the vhb gene was transferred into pBin19 for genetic transformation of white poplar (Populus alba L.) via Agrobacterium tumefaciens. Southern blot analysis confirmed the presence of vhb sequence in poplar genome. Accumulation of vhb transcript was detected in transgenic leaf tissue by Northern blot analysis. In vitro growth measurements showed that vhb gene expression in transgenic poplar plants did not significantly affect their growth patterns, including stem elongation, leaf enlargement, and shoot and root biomass. Transgenic VHb poplar lines displayed different levels of growth and biomass production in the greenhouse. Two of the selected transgenic lines showed significantly higher values for stem diameter, number of stem nodes and internodes, and exhibited enhancement of growth and biomass production when compared to the control plants. Transgenic plants showed higher chlorophyll a, b, and total protein contents compared to wild-type plants. In contrast, there were also two transgenic lines that revealed plant growth performances in greenhouse significantly lower than the control plants. Our results show that VHb expression in transgenic poplar can have erratic effects since it did not always result into enhancement of plant growth and biomass production. However, the promising results reported for some of the transgenic poplar lines and other plant species strongly indicate the potential future application of this approach to enhance these commercially important traits in breeding programs.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.28

HAIRY ROOT INDUCTION IN HELICHRYSUM ITALICUM AND H. STOECHAS FOR THE PRODUCTION OF SECONDARY METABOLITES

M. AMORETTI, A. DI GUARDO, P. FILOTTI, B. RUFFONI, A. GIOVANNINI 

Istituto Sperimentale per la Floricoltura, Corso degli Inglesi 508, 18038 Sanremo (Imperia)

Agrobacterium rhizogenes, flavonoids, everlasting, H. angustifolium, no food plants 

Helichrysum italicum (Roth) G. Don and H. stoechas (L.) Moench are two medicinal species of the Mediterranean region, belonging to the genus Helichrysum (family Asteraceae). The plants are evergreen shrubs growing in arid soils, from the cliffs above the sea to the hills, where they flower from May to August. The bright yellow flower heads contain principally essential oils, flavonoids (helichrysin A and B) and tannins. H. italicum and H. stoechas have been used in folk medicine because of their antibacterial, antitoxic, diuretic and antiallergic properties; moreover the flowering stems are dried and used as "everlasting flowers". The neoplastic roots (hairy roots), induced by the soil Gram negative bacterium Agrobacterium rhizogenes, are a biosynthetically active tissue that can express root-specific metabolic pathways as efficiently as intact plant. The secondary metabolite synthesis is not strictly limited to those that are normally produced in roots of differentiated plants (Flores et al., Plant Physiol., 1993, 101: 363-371). 

We report a protocol for hairy root induction in H. italicum and H. stoechas. Leaf and root tissues of micropropagated plant clones (Giovannini et al., Acta Horticolturae, in press) were co-cultivated with a solution of A. rhizogenes wild type strain 15834 (ATCC). After 3 days the explants were transferred to a medium without growth regulators, containing basal salts and vitamines MS (Murashige and Skoog, Physiol. Plant., 1962, 15: 473-497), 30 g/l sucrose and 8 g/l agar (Basal Medium), supplemented with Cefotaxime (100 mg/l). The number of explants developing roots was counted after 30, 60 and 90 days. One hundred percentages of root explants developed new roots after 30 days of culture, in the two species. After 90 days, new roots were observed in 92,3% of H. italicum and in 18,7% H. stoechas leaf explants. Several putative hairy roots were isolated and further propagated on Basal Medium. PCR analysis was performed on root total genomic DNA, using specific primers for the amplification of A. rhizogenes rolC and virC1 genes. A rolC gene amplification band of 600bp was detected in 3 putative H. italicum hairy root lines; virC1 amplification band of 326 bp was visualised only in the bacterium sample. H. italicum hairy root cultures were established in liquid Basal Medium and were cultivated in light and dark conditions, in continuous shaking. The pH of the medium was monitored for five-week culture. 

H. italicum hairy root culture is a fast growing active tissue that can be exploited for the production of secondary metabolites and for the scaling up in bioreactor.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.29

ANALYSIS OF ESSENTIAL OIL PRODUCTION IN VETIVERIA ZIZANIOIDES (L.) NASH DURING EARLY GROWTH

D.R. MASSARDO*, M. CAPUANO**, F. SENATORE**, L. DEL GIUDICE*

*) Istituto di Genetica e Biofisica “Adriano Buzzati-Traverso” – CNR, via G. Marconi 10, 80125 Napoli
**) Dip.to Chimica delle Sostanze Naturali, Università “Federico II”, via D. Montesano 49, 80131 Napoli

Vetiver oil, gas chromatography/mass, khusimone, khusimol, symbiotic bacteria

The species Vetiveria zizanioides (L.) Nash (Gramineae) is known for its ability to produce odorous roots, which can be used for the extraction of an essential oil of great economic importance (Maffei 2002). The essential oil that is produced in secretory cells located inside the mature roots has been used since ancient times as a perfume and a natural remedy against human and animal disease. It consists of a complex mixture of sesquiterpene hydrocarbons and alcohols, which are mostly used as a basic material for perfurmery.

Recently has been reported the electron microscope analysis of vetiver cells of the roots, evidencing the presence of bacteria into lysigen lacunae, which are cellular structure of the vetiver roots in which is stored the essential oil (Maffei 2002). The close relationship between those bacteria and the essential oil stimulated the idea of a direct involvement of those symbiotic bacteria in the essential oil metabolism.

As a preliminary work, in a tentative to gain on insight to essential oil metabolism we have analyzed the oil production in vetiver during early growth.

Planting of Vetiveria zizanioides in Campania region was performed in the spring using vetiver culmus with short roots and with approximately 20 cm of leaves. Root samples were collected at intervals of two months starting from transplantation. The freshly collected roots were cut into small pieces, then hydrodistilled for 3 hours according to the standard procedure as described in the European Pharmacopoeia (1997), using a Clevenger-type apparatus to produce oil. The essential oil was analyzed by GC and GC/MS. GC analyses were performed on a Perkin-Elmer Sigma-115 gas chromatograph equipped with a DB-5 fused-silica column, injector and detector temperatures 250°C and 285°C, respectively; He as carrier gas. CG/MS analysis was performed using a Hewlett-Packared 5890A apparatus,equipped with a HP-1 fused-silica column, linked on line with a HP Mass Selective Detector (MSD 5970 HP); ionization voltage 70eV; electron multiplier energy 2000 V.

The production of vetiver oil was constant, 0,30 g oil/100 g roots, during the first 6 months (from May to October) growth from transplantation. During 8 months growth there was a doubling of production of essential oil, 0,70 g oil/100 g roots, before starting in turn a continuous decrease of oil production: during 10 months growth it was  0,6 g oil/100 g. roots; while during 12 months growth it was  0,30 g oil/100 g roots. Besides, the analysis of two principal vetiver oil costituents, the tricyclic sesquiterpenes khusimone and khusimol has shown that both the latter components maintain costant their rate percent in the vetiver oil during the growth from transplantation. 

Our data suggest that vetiver oil production is closely related to the metabolism of plant which is affected by changes in environmental temperatures (Maffei 2002). During winter time the drop in temperature causes a decrease of plant metabolic activities which in turn slow down oil production. 

It could suppose that also symbiotic bacteria are affected by changes in environmental temperatures so that their activity in metabolizing vetiver essential oil is related to the modulation of plant metabolism.

Work is in progress to isolate symbiotic bacteria from vetiver roots. 

References
- M. Maffei (2002). Introduction to the genus Vetiveria. In “Vetiveria . The genus Vetiveria” (ed. M. Maffei), 1-18. Taylor & Francis, London and New York.
- European Pharmacopoeia 3th Ed., p. 121, Council of Europe, Strasbourg (1997)

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 3.30

TRASCRIPTION PROFILE OF GENES INDUCED BY CADMIUM IN BRASSICA JUNCEA FOR PHYTOREMEDIATION

N. FUSCO, G. DAL CORSO, L. BORGATO, A. FURINI

Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada Le Grazie 15, 37134 Verona

Heavy metal contamination of soil poses serious problems to both human health and agriculture. The available engineering-based technologies used to remediate soils are quite costly, and often dramatically disturb the landscape. Recently, there has been considerable interest focused on the use of several terrestrial plants able to absorb heavy metals from the soil and concentrate them in the harvestable shoot tissues as an alternative remediation technology. These plants have developed networks that enable them to regulate the uptake, the translocation and sequestration of heavy metals to limit the exposure of cytosolic proteins to toxic elements such as cadmium and lead. Up to now most components of this network remain unidentified and little is known about the signal transduction cascades involved in gene regulation due to changes in the metal status of soils. Therefore, to molecularly characterize these processes we chose Brassica juncea as a model plant for accumulation and tolerance of heavy metals. Using the cDNA-AFLP expression profiling we are studying transcriptional changes upon exposure to Cd for 6 h, 24 h and 6 weeks. So far 130 fragments of consistently up-regulated, transiently regulated and down-regulated signal have been isolated and sequenced. Several putative signal transduction components such as a bZIP transcriptional factor, a putative receptor protein kinase, a nodA protein, a phenylalanine ammonia liase and several proteins with unknown function have been already identified. These data will help to better understand the process of Cd accumulation and tolerance. Eventually, this information will be used to develop more effective remediating plant species and agronomic practices that enhance phytoremediation.