Plant regeneration from an endangered valuable cork oak tree by somatic embryogenesis.

Among the several applications of in vitro tissue culture techniques, the conservation of plant germplasm is one of the most widely used. The cork oak is one of the principal tree species in the Western Mediterranean región. Within this área, the Balearic Islands are considered to be a glacial refuge, and therefore a reservoir of genetic resources. A singular tree has been found in the small Minorca Island population. The haplotype of this tree is of Tyrrhenian origin, showing a past link between Minorca and Sardinia. Moreover, this tree do not bear a deletion within an ITS from ribosimic nuclear DNA, which is fairly common in many populations of this species, and indicates that ir may be the descendant of a very ancient population. This tree is currently in a precarious condition, and it has not produced acorns in the last years. Hence there is a clear need of vegetative propagation to conserve this genotype. We have previously developed methods to clone adult cork oak tres by somatic embryogenesis, and therefore the aim of the present work was to clone this singular tree. There braches from the corwn were collected in November 2004, and methods previously described were carried out. By February 2005 somatic embryogenesis was obtained from leaves of the tree with percentages on induction ranging from 17 to 54% depending on the branch, which may show a novel source of variation that requires further study. Spontaneously matured somatic embryos germinated at 46% in average, and the first somatic seedlings from the Alfavaret's cork oak tree were obtained. Therefore, this study shows one of the most relevant applications of somatic embryogenesis: the plant regeneration of valuable genotypes for the in situ and ex situ conservation of forest genetic resources.

Purification and Characterization of AsES Protein A Subtilisin Secreted by Acremonium Strictum is a Novel Plant Defense Elicitor.

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

Does the natural "microcosm" created by Tuber aestivum affect soil microarthropods? A new hypothesis based on Collembola in truffle culture

microarthropods play an important role in fungi dispersion, but little is still known about the interaction between truffle and soil microarthropods. The aim of this study was to investigate the ability of the truffle Tuber aestivum to modify soil biogeochemistry (i.e. create a zone of scarce vegetation around the host plant, called a burn or brûlé) and to highlight the effects of the brûlé on the soil fauna community. We compared soil microarthropod communities found in the soil inside versus outside the T. aestivum brûlé with the chemistry of soil collected inside versus outside the brûlé. The study was carried out in three Mediterranean areas, two in Italy and one in Spain. The results confirmed the ability of T. aestivum to modify soil biogeochemistry in the brûlé: pH was higher and total organic carbon tended to be lower inside the brûlé compared to outside. Soil fauna communities showed some interesting differences. Some groups, such as Symphyla and Pauropoda, adapted well to the soil; some Collembolan families, and biodiversity and soil quality indices were generally higher outside the brûlé. Folsomia sp. showed higher abundance in the soil of the brûlé compared to outside. The results suggest that some Collembola groups may be attracted by the fungal metabolites produced by T. aestivum, while other Collembola and other microarthropods may find an unfavourable environment in the soil of the brûlé. The next steps will be to confirm this hypothesis and to extend the study to other keys groups such as nematodes and earthworms and to link fluctuations of soil communities with the biological phases of truffle growth.

Post-embryonic organogenesis and plant regeneration from tissues: two sides of the same coin?

Plants have extraordinary developmental plasticity as they continuously form organs during post-embryonic development. In addition they may regenerate organs upon in vitro hormonal induction. Advances in the field of plant regeneration show that the first steps of de novo organogenesis through in vitro culture in hormone containing media (via formation of a proliferating mass of cells or callus) require root post-embryonic developmental programs as well as regulators of auxin and cytokinin signaling pathways. We review how hormonal regulation is delivered during lateral root initiation and callus formation. Implications in reprograming, cell fate and pluripotency acquisition are discussed. Finally, we analyze the function of cell cycle regulators and connections with epigenetic regulation. Future work dissecting plant organogenesis driven by both endogenous and exogenous cues (upon hormonal induction) may reveal new paradigms of common regulation.