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Several dicotyledonous species were infected with an Agrobacterium rhizogenes binary vector harbouring the plasmid 121.Sn which contains the maize gene Sn under the constitutive promoter CaMV35S. In maize, Sn transactivates the anthocyanin pathway in different tissues. The aim of this work was to test the efficiency of this gene to regulate the anthocyanin pathway in heterologous systems and verify its suitability as a reporter gene. The pigmentation of the hairy roots was compared with hairy roots stained for β-glucuronidase activity, which were used as a control. In two polymorphic genotypes of Lotus angustissimus, DNA integration and expression were assayed. The maize gene is competent to induce anthocyanin pigmentation in different species, but the complexity of the regulatory mechanisms of anthocyanin synthesis restricts the use of Sn as a reporter gene.

Saponins, a group of glycosidic compounds present in several plant species, have aglycone moieties that are formed using triterpenoid or steroidal skeletons. In spite of their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still poorly understood. In the Medicago genus, the hemolytic activity of saponins is related to the nature of their aglycone moieties. We have identified a cytochrome P450 gene (CYP716A12) involved in saponin synthesis in Medicago truncatula using a combined genetic and biochemical approach. Genetic loss-of-function analysis and complementation studies showed that CYP716A12 is responsible for an early step in the saponin biosynthetic pathway. Mutants in CYP716A12 were unable to produce hemolytic saponins and only synthetized soyasaponins, and were thus named lacking hemolytic activity (lha). In vitro enzymatic activity assays indicate that CYP716A12 catalyzes the oxidation of β-amyrin and erythrodiol at the C-28 position, yielding oleanolic acid. Transcriptome changes in the lha mutant showed a modulation in the main steps of triterpenic saponin biosynthetic pathway: squalene cyclization, β-amyrin oxidation, and glycosylation. The analysis of CYP716A12 expression in planta is reported together with the sapogenin content in different tissues and stages. This article provides evidence for CYP716A12 being a key gene in hemolytic saponin biosynthesis.

Creeping-rootedness (CR) is a form of vegetative propagation in lucerne (Medicago sativa L. complex) consisting of the development of new plants from adventitious shoots arising from roots with horizontal growth habit. Introgression of the CR character into cultivated lucerne may contribute to better stand persistence and greater grazing tolerance. Phenotypic selection of this trait is difficult owing to intrinsic features of the species such as autotetraploidy, allogamy and propagation by seed in addition to the polygenic control, low espressivity and variable penetrance of the trait. With the aim of developing a molecular marker-based system for the genotypic selection of the CR phenotype in lucerne, two segregating populations for this trait were screened with amplified fragment length polymorphisms (AFLPs), in order to identify CR-linked markers. Two AFLP amplicons were selected as segregating in a non-independent manner from the CR phenotype in both populations. The use of AFLP closer to the CR allows the conditional probability for CR selection to be nearly doubled. A PCR-based marker was developed for this AFLP and used to screen a collection of genotypes displaying either the CR or non-CR phenotype. The related primers were able to amplify the diagnostic amplicon in most of the CR genotypes and in approximately half of the non-CR individuals. Results are discussed in relation to the use of this marker in marker-assisted selection of the CR phenotype in lucerne.

Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis.

Anthocyanins and condensed tannins are major flavonoid end-products in higher plants. While the transactivation of anthocyanins by basic helix-loop-helix (bHLH) transcription factors is well documented, very little is known about the transregulation of the pathway to condensed tannins. The present study analyses the effect of over-expressing an Sn transgene in Lotus corniculatus, a model legume, with the aim of studying the regulation of anthocyanin and tannin end-products. Contrary to expectation, effects on anthocyanin accumulation were subtle and restricted to the leaf midrib, leaf base and petiole tissues. However, the accumulation of condensed tannin polymers was dramatically enhanced in the leaf blade and this increase was accompanied by a 50-fold increase in the number of tannin-containing cells in this tissue. A detailed analysis of selected lines indicated that this transactivational phenotype correlated with high steady-state transcript levels of the introduced transgene and the introduction of a single copy of the CaMV35S-Sn construct into these clonal genotypes. While the levels of condensed tannins in leaves were increased by up to 1% of the dry weight, other major secondary end-products (flavonols, lignins and inducible phytoalexins) were unaltered in transactivated lines. These results give an initial insight into the developmental and higher-order regulation of polyphenolic metabolism in Lotus and other higher plant species.

We have established mutant collections of the model species Medicago truncatula according to current protocols. In particular, we used a transposon (Tnt1) tagging method and an ethyl methanesulfonate (EMS) mutagenesis approach (TILLING). The collections were subjected to both forward and reverse genetics screenings, and several mutants were isolated that affect plant traits (e.g. shoot, root developments, flower morphology, etc.) and also biosynthetic pathways of secondary compounds (saponins and tannins). Genes responsible for some of the mutations were cloned and further characterized.

Three seed lots, obtained from different 'foundation farms', for each of the alfalfa, Medicdgo saliva L., ecotypes 'Vogherese' and 'Maremmana', together with the variety 'Iside' as a control, were studied to test the possibility of distinguishing them through restriction fragment length polymorphism analysis. Twenty plants per seed lot were analysed with 25 DNA probes that yielded 155 informative polymorphic fragments. Variance partitioning showed that within-population variability accounted for nearly 98% of the total variance, while a small but significant contribution to the total variance was due to among-lot variability. 'Vogherese' was more homogeneous than 'Maremmana', as a consequence of the greater environmental homogeneity of the adaptation area of the former ecotype compared with the latter. Bulk analysis yielded eight variety-specific bands, 12 bands present in both ecotypes and absent in the variety and one band specific of 'Maremmana'. Results are discussed in relation to the practical use of molecular markers for alfalfa ecotype identification.

Accumulation of zeins, the endosperm storage proteins of maize, in a heterologous plant expression system was attempted. Plants of Nicotiana tabacum and Lotus corniculatus were transformed by Agrobacterium with binary vectors harbouring genes that code for γ-zein and β-zein, two zeins rich in sulphur amino acids. Adding the ER retention signal KDEL to the C-terminal domain modified the zein polypeptides. Significant levels of γ-zein:KDEL and β-zein:KDEL were detected in primary transformants of tobacco. Moreover, the two zeins colocalized in leaf protein bodies of γ-/β-zein:KDEL plants derived from a cross between two primary transformants. Coexpression of γ-zein:KDEL and β-zein:KDEL could be a useful strategy to obtain genotypes of forage legumes which are able to accumulate sulphur amino acids to high levels. As a first step, L. corniculatus plants expressing γ-zein:KDEL in the leaves were obtained.

A cytological examination of the nucleolus organizing regions (NORs) of three species from the Medicago sativa complex was conducted to evaluate the structural and functional evolution of the ribosomal RNA (rRNA) loci that encode the 18S, 5.8S, and 26S rRNAs. Mitotic chromosomes in root-tip preparations from tetraploid M. sativa and diploids Medicago coerulea and Medicago falcata were visualized by four methods that provide new data. Fluorescent in situ hybridization using the M. sativa 18S gene as probe localized the structural rDNA to the constricted regions of the satellited chromosomes only. Chromomycin A3 (CMA3) staining and 4′,6-diamidino-2-phenylindole (DAPI) staining identified these chromosomal segments as the most GC-rich regions in the alfalfa karyotype. Medicago falcata exhibited fewer DAPI bands and chromocenters than did M. sativa and M. coerulea. Positive silver nitrate staining showed that all four rDNA regions in M. sativa (located in two chromosome pairs) and both rDNA sites in both diploid species remain transcriptionally active. Counts of nucleoli confirmed that all rDNA regions are independently capable of nucleolus organization. Thus, the number of active NORs in M. sativa is double the number found in M. coerulea or M. falcata. Consequently, if M. sativa originated from sexual hybridization of 2n gametes involving one or both diploid species, no major reorganization or loss of structural or functional rDNA loci has occurred. Key words : alfalfa evolution, CMA3 banding, DAPI banding, fluorescent in situ hybridization, silver nitrate staining.

Culture conditions have been established for callus induction and growth from different explants in L. angustissimus L. Calli were obtained from hypocotyls, leaves, stems, cotyledons and roots cultured on media containing 2,4-dichlorophenoxyacetic acid or α-naphthaleneacetic acid with kinetin, N6 − Δ2 or benzyladenine in different combinations and concentrations. Only those calli induced in presence of α-naphthaleneacetic acid with benzyladenine or kinetin produced shoots. Calli induced from hypocotyl explants were the most efficient in regeneration of shoots. Transformation with an Agrobacterium rhizogenes binary vector carrying the plasmid pBI 121.1 is reported. The percentage of cotransformation was estimated by testing GUS activity in hairy roots. The integration of Ri T-DNA and the NPTII gene in transformed plants was confirmed by molecular analyses and in vitro culture of transgenic tissues in the presence of kanamycin.

Plant regeneration from explant and protoplast derived callus has been achieved in Medicago littoralis cv. Harbinger 1886, an annual legume resistant to the fungus Pseudopeziza medicaginis. Callus was induced from different tissue explants and the fastest growth rate was observed for hypocotyls in B5 medium with 2 mg l−1 2,4-dichlorophenoxyacetic acid and 0.5 mg l−1 N6-benzyladenine. Protoplasts were isolated from cotyledons and leaves of sterile plants and from callus; the first two kinds of protoplasts showed a plating efficiency of 5.6% and 5%, respectively, when embedded in agarose. Plant regeneration occurred on media containing % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9qq-f0-yqaqVeLsFr0-vr% 0-vr0db8meaabaqaciGacaGaaeqabaWaaeaaeaaakeaacaqGobWaaW% baaSqabeaacaqG2aaaaOGaaeOVfiaabs5adaahaaWcbeqaaiaaikda% aaGccaqG+waaaa!3F97!\[{\text{N}}^{\text{6}} {\text{\Delta }}^2 {\text{}}\]isopentenyl-adenine combined with indole-3-acetic acid or 1,2-benzisoxazole-3-acetic acid, and on media with N6-benzyladenine plus α-naphtaleneacetic acid; a cytokinin/auxin ratio higher than 1 induced embryos while a ratio around 1 stimulated shoot formation. Embryo development and rooting of shoots were performed in RL medium without growth regulators.

Proanthocyanidins (PAs) are agronomically important biopolymers in higher plants composed primarily of catechin and epicatechin units. The biosynthesis of these natural products is regulated by transcription factors including proteins of the R2R3MYB class. To gain insight into the genetic control of the catechin and epicatechin branches of the PA pathway in forage legumes, here the effects of the expression of FaMYB1, a flavonoid R2R3MYB repressor from strawberry, in Lotus corniculatus (birdsfoot trefoil), were tested. It was found that in leaves of T(0) transgenic lines the degree of PA inhibition correlated with the level of FaMYB1 expression. These effects were heritable in the transgene-positive plant T(1) generation and were tissue specific as the suppression of proanthocyanidin biosynthesis was most pronounced in mesophyll cells within the leaf, whereas other flavonoid and phenolic compounds were substantially unaltered. The data suggest that FaMYB1 may counter-balance the activity of the endogenous transcriptional MYB-bHLH-WD40 (MBW) complex promoting proanthocyanidin biosynthesis via the catechin and epicatechin branches and that FaMYB1 does not interfere with the expression levels of a resident R2R3MYB activator of PAs. It is proposed that in forage legumes leaf cell commitment to synthesize proanthocyanidins relies on the balance between the activity of activator and repressor MYBs operating within the MBW complex.

Nodes ofMedicago sativa, Lotus corniculatus, Lotus tenuis, andLotus pedunculatus were cultured on MS basal media with different growth regulators. InM. sativa each node produced one shoot and the apical dominance was unaffected by high levels of cytokinins, and subsequent cycles of culture. Shoot development was stimulated by the presence ofN 6-isopentenyl-adenine in the culture medium and was dependent on the genotype of the explant. Shoot development was not affected by the original position of the node on the plant nor by the plant age. Shoots rooted in MS medium gelled with starch and containing 2 mg·liter−1 indol-3-acetic acid. In the threeLotus species, node culture was a more effective technique than inM. sativa. The number of shoots per node increased with the time of culture and with the presence of 0.05 mg·liter−1 of 6-benzylaminopurine. The highest number of shoots derived from one node was achieved inL. pedunculatus and inL. tenuis by culturing single nodes, whereas inL. corniculatus stem segments had to be totally covered by the medium for success. Rooting was easily achieved in MS medium with or without auxins.

Protein bodies (PB) are stable oligomers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. We have tried to promote the formation of Nef-containing protein bodies in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein gamma-zein or to the chimeric protein zeolin (which efficiently forms protein bodies and is composed of the vacuolar storage protein phaseolin fused to the Nterminal domains of gamma-zein). Protein blots and pulse-chase indicate that fusions between Nef and the same gamma-zein domains present in zeolin are degraded by ER quality control. Coherently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin-Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. We conclude that zeolin, but not its gamma-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants.

Plant regeneration from leaf- and cotyledon-derived calli and from protoplast-derived tissue has been obtained in Lotus pedunculatus. Callus induction was achieved with 2,4-D and plant regeneration required the following two media sequences: bud formation was stimulated by IAA and BA and shoot growth by kinetin. Root formation occurred in the presence of IAA. Cotyledon protoplasts showed a low plating efficiency and plant regeneration was achieved via an intervening callus phase.

Lucerne (alfalfa —Medicago sativa) pollen, cultured at the late unicellular stage, followed one of two developmental pathways: (1) A pathway involving symmetric mitosis which produces pollen containing two vegetative (2 V) or two generative (2 G) pollen. This morphology was only observed in culture, and pollen which followed this developmental path is defined as non-physiological. Occasionally the formation of multi-nucleate pollen grains containing from 4–9 cells were observed. Sustained divisions were not observed. (2) The production of bicellular (V+G) pollen followed by tricellular (V+2G) pollen. Since these types of grains are encountered during development in vivo, pollen following this developmental pathway is defined as physiological. The proportion of pollen that divided was enhanced by a cold treatment at 4°C for one week, prior to culture. The ratio of non-physiological (i.e., 2V or 2G) to physiological pollen (G+V or 2G+V) was found to be affected by the nature of the osmoticum in the medium. Media containing maltose or melibiose gave higher proportions of non-physiological pollen than media containing glucose or sucrose.

Several strategies have been exploited to maximize heterologous protein accumulation in the plant cell. Recently, it has been shown that a portion of a maize prolamin storage protein, gamma-zein, can be used for the high accumulation of a recombinant protein in novel endoplasmic reticulum (ER)-derived protein bodies of vegetative tissues. In this study, we investigate whether this protein can be expressed in the chloroplast. Our long-term purpose is to use zeolin to produce value-added proteins by fusing these polypeptides with its gamma-zein portion and targeting the recombinant proteins to the ER or to the chloroplast. We show here that zeolin accumulates in the chloroplast to lower levels than in the ER and its stability is compromised by chloroplast proteolytic activity. Co-localization of zeolin and the ER chaperone BiP in the chloroplast does not have a beneficial effect on zeolin accumulation. In this organelle, zeolin is not stored in protein bodies, nor do zeolin polypeptides seem to be linked by inter-chain disulfide bonds, which are usually formed by the six cysteine of the gamma-zein portion, indicating abnormal folding of the recombinant protein. Therefore, it is concluded that to accumulate zeolin in the chloroplast it is necessary to facilitate inter-chain disulfide bond formation.

Zeins, the main storage proteins of maize that accumulate in the endoplasmic reticulum of the endosperm cells, are particularly interesting because they are rich in the essential sulphur amino acids. Overexpression of certain zein genes in plants such as alfalfa would be expected to improve the nutritional characteristics of this crop. Recently, significant accumulation values have been reached, but still far from those considered useful for nutritional purposes. This study investigates whether targeting to compartments other than the endoplasmic reticulum (cytosol and chloroplasts) could result in increasing beta-zein accumulation in transgenic plants. To address beta-zein to the cytosol, the fragment which codes for the signal peptide has been removed. beta-zein has also been targeted to alfalfa and tobacco chloroplasts by a transit peptide signal. Both tobacco, as a model plant species, and alfalfa have been transformed with the assembled constructs. An alternative route to accumulate beta-zein in the chloroplasts is to synthesize beta-zein directly in the plastid lumen. Thus, the beta-zein gene has also been inserted into tobacco plastid DNA. The beta-zein gene in each different type of transformed plant was properly transcribed, as determined by northern blot analysis, but no accumulation of beta-zein was detected, either in the cytoplasm or in the chloroplasts of alfalfa and tobacco transformed plants. Therefore, it is concluded that chloroplasts and the cytosol are not favourable subcellular locations for zein protein accumulation.

The major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris), zein and phaseolin, accumulate in the endoplasmic reticulum (ER) and in storage vacuoles, respectively. We show here that a chimeric protein composed of phaseolin and 89 amino acids of γ-zein, including the repeated and the Pro-rich domains, maintains the main characteristics of wild-type γ-zein: It is insoluble unless its disulfide bonds are reduced and forms ER-located protein bodies. Unlike wild-type phaseolin, the protein, which we called zeolin, accumulates to very high amounts in leaves of transgenic tobacco (Nicotiana tabacum). A relevant proportion of the ER chaperone BiP is associated with zeolin protein bodies in an ATP-sensitive fashion. Pulse-chase labeling confirms the high affinity of BiP to insoluble zeolin but indicates that, unlike structurally defective proteins that also extensively interact with BiP, zeolin is highly stable. We conclude that the γ-zein portion is sufficient to induce the formation of protein bodies also when fused to another protein. Because the storage proteins of cereals and legumes nutritionally complement each other, zeolin can be used as a starting point to produce nutritionally balanced and highly stable chimeric storage proteins.

Evidence from numerous feeding studies with cows indicates that excessive ruminal protein degradation may be the most limiting nutritional factor in higher-quality temperature legume forages such as alfalfa and clover (Barry and McNabb, 1999). Condensed tannins (CT), found in some minor forage legumes, are known to decrease protein degradation. However, genes involved in CT biosynthesis are still largely unidentified. Since Lotus corniculatus is an in vitro highly responsive species and it does accumulate CT in edible tissues, it represents an invaluable legume model species to gain information on regulatory mechanisms leading to CT biosynthesis.

Forage legumes are essential components in the diet of cattle and sheep unless they are fed with rendered animal proteins, which may have consequent serious problems for animal and human health. The most cultivated forage species are lucerne and clovers. However, the large amounts of proteins available from these species are poorly exploited, since they are degraded extensively both by plant proteases and rumen microorganisms where N may be lost through NH3 formation. These degradative phenomena are the basis of a range of different and serious agricultural problems: 1) bloating with consequent risks for the health and the life of animals;1 2) loss of nutritive value and reduced growth of the animals;2 3) pollution of the environment; in fact, excreted ammonia-rich manure is a pollution factor that alters ecological equilibrium and the environment.3

Species-specific internal ITS primers that amplify polymerase chain reaction (PCR) products of different lengths were selected to distinguish the morphologically similar ectomycorrhizal fungi T. melanosporum, T. brumale and T. indicum by aligning their internal transcribed spacer sequences and taking into account any incidence of intraspecific variability. In multiplex PCR experiments, the species-specific primers yielded the expected amplicons on template DNA isolated from the above mentioned species, while there was no amplification in PCR reactions carried out on fungal DNA from competing truffle species and host plants.

Methods were developed to type some of the most economically important Tuber spp. (Tuber aestivum f. uncinatum, Tuber albidum, Tuber magnatum, Tuber brumale, Tuber melanosporum) and some of their mycorrhizae by the use of molecular markers. The polymorphism of the rDNA region was investigated by polymerase chain reaction (PCR) and Southern experiments on DNA isolated from ascocarps, uninoculated host roots and mycorrhizae. Using specific primer pairs, PCR amplification of the internal transcribed spacers (ITS) and intergenic spacer (IGS) of DNA isolated from fruitbodies allowed fungus discrimination only by the length or the number of the ITS and IGS amplified fragments. These results were confirmed by Southern analysis carried out on fungus DNA restricted with EcoRI and probed with the ITS fragment of T. brumale: a specific banding pattern for each species tested was evident. The same analyses were then performed on mycorrhizal root tips and inoculated host plant roots. Each fungus-host plant interaction showed a specific pattern due to the combination of traits of the two partners. It is concluded that rDNA region is a suitable molecular marker to characterize both ascocarps and mycorrhizae of truffle species by PCR and/or Southern analysis.

Androgenic plants have been obtained via anther culture in four natural populations of Hordeum spontaneum. Microscopic observations revealed that androgenesis started with the formation of two vegetative-type nuclei derived from the mitotic division of the uninucleate microspores. In this species androgenesis was affected by the type and concentration of the sugars added to the culture medium: the highest response (17% of callusing anthers) was observed on media containing 80 g l(-1) maltose. The highest production of androgenic plants (per 100 anthers, 5.9 green and 4.3 albino plants) was obtained from callus grown on these same media. About half of the green plants regenerated were haploid, while the others were diploid and set seed.

Lotus corniculatus L. plants were transformed with Agrobacterium rhizogenes binary vector carrying the maize Sn regulatory gene driven by the 35S promoter. These plants showed modifications in the pattern of accumulation of proanthocyanidin (PA). All the transformed plants but one showed an increase in PA content in the root relative to control untransformed and control gus gene transformed plants (C). With respect to the PAaccumulation in leaves, Sn transgenic plants were grouped in two classes: suppressed (S), that showed a consistent reduction of foliar PAcontent, and unsuppressed (U) that did not differ significantly from controls. Dihydroflavanol reductase (DFR) and leucocyanidin reductase (LAR) enzyme activities in S and U plant leaves mirrored the changes seen with foliar PA accumulation. LAR activity in the roots was consistent with the root PA levels. Mature Sn mRNA accumulated in the leaves of U plants, but not in leaves of S plants; however, leaves of both S and U plants were able to initiate Sn transcription. All Sn-transformed plants accumulated Sn message in root tissue. A possible negative interaction of Sn and an unidentified homologous endogene is proposed for explaining the behaviour of S plants.

Culture conditions have been established for callus induction and growth from different explants in Lotus tenuis . Fast-growing calli from cotyledons, roots and hypocotyls were obtained on media containing 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalenacetic acid (NAA) alone. With leaves, best results were achieved with 2,4-D plus 6-benzylaminopurine (6-BAP) or kinetin. Only those calli grown in the presence of NAA with 6-BAP or kinetin produced shoots when transferred to regeneration medium. Protoplasts were obtained from cotyledons, roots and leaves, but only cotyledon and root protoplasts were able to divide. Plant regeneration was obtained from all kinds of calli.

PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes.