Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Analysis of PEAM4, the pea AP1 functional homologue, supports a model for AP1-like genes controlling both floral meristem and floral organ identity in different plant species.

APETALA1 (AP1) and its homologue SQUAMOSA (SQUA) are key regulatory genes specifying floral meristem identity in the model plants Arabidopsis and Antirrhinum. Despite many similarities in their sequence, expression and functions, only AP1 appears to have the additional role of specifying sepal and petal identity. No true AP1/SQUA-functional homologues from any other plant species have been functionally studied in detail, therefore the question of how the different functions of AP1-like genes are conserved between species has not been addressed. We have isolated and characterized PEAM4, the AP1/SQUA-functional homologue from pea, a plant with a different floral morphology and inflorescence architecture to that of Arabidopsis or Antirrhinum. PEAM4 encodes for a polypeptide 76% identical to AP1, but lacks the C-terminal prenylation motif, common to AP1 and SQUA, that has been suggested to control the activity of AP1. Nevertheless, constitutive expression of PEAM4 caused early flowering in tobacco and Arabidopsis. In Arabidopsis, PEAM4 also caused inflorescence-to-flower transformations similar to constitutive AP1 expression, and was able to rescue the floral organ defects of the strong ap1-1 mutant. Our results suggest that the control of both floral meristem and floral organ identity by AP1 is not restricted to Arabidopsis, but is extended to species with diverse floral morphologies, such as pea.

Isolation, culture and division of olive (Olea europaea L.) protoplasts.

Protoplasts from Olea europaea L. have been compared in terms of their yield, viability, cell division and callus differentiation. Viable protoplasts were isolated from in vitro cultured leaves and cotyledons by an overnight incubation in an enzyme solution containing 1-1.5% driselase and 0.5M sucrose. This method allowed high yield of purified protoplasts, which floated and formed a dark green band at the meniscus, after centrifugation. Purified protoplasts were diluted to 3×10(4) protoplasts·ml(-1) in culture medium. After cell wall regeneration, protoplasts gradually increased their volumes under appropriate conditions. The first divisions occurred during the second week in culture. Division efficiency ranged from 5.2 to 9.8% after 20 days in culture. Two weeks later visible microcolonies developed only from cotyledon protoplasts. After 6 weeks in culture, the microcalli were transferred to a solidified culture medium with 0.6% agarose, which induced active callus growth.