An alternative method for transforming sweet orange [Citrus sinensis (L.) Osbeck] has been developed. Plasmid DNA encoding the non-destructive selectable marker enhanced green fluorescent protein gene was introduced using polyethylene glycol into protoplasts of`Itaborai' sweet orange isolated from an embryogenic nucellar-derived suspension culture. Following protoplast culture in liquid medium and transfer to solid medium, transformed calluses were identified via expression of the green fluorescent protein, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. Transgenic plantlets were recovered from germinating somatic embryos and by in vitro rooting of shoots. To expedite transgenic plant recovery, regenerated shoots were also micrografted onto sour orange seedling rootstocks. Presence of the transgene in calluses and regenerated sweet orange plants was verified by gene amplification and Southern analyses. Potential advantages of this transformation system over the commonly used Agrobacterium methods for citrus are discussed.

Microsporogenesis was analysed in a tetraploid somatic hybrid (SH) (2n 4x 36) of Citrus and its diploid fusion parents (2n 2x 18), Valencia sweet orange (C. sinensis L. Osbeck) and Femminello lemon (C. limon L. Burm. f.). Intergenomic pairing between lemon and orange occurred in the somatic hybrid which showed multivalent chromosome associations in diakinesis, although one quadrivalent was de®nitely because of a reciprocal translocation present in Valencia. The behaviour of univalents was variable in the somatic hybrid and its parents. In the somatic hybrid and Valencia, the univalents preferentially formed micronuclei and polyads whereas, in Femminello, they were generally enclosed in a nucleus although distributed randomly. The somatic hybrid showed a rate of pollen stainability of 64% and germinability of 41%. 1The chromosomally unbalanced pollen from the tetraploid SH was presumed viable and able to fertilize because di erent nuclear DNA contents were found in the back-cross progeny. Moreover, meiotic nuclear restitution mechanisms, which could be mainly dependent on the abnormal orientation of the spindles in meiosis II, are described.

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Plants produce considerable amounts of volatile organic compounds (VOCs) with several biological functions, including protection against biotic agents such as viruses and their vectors. In citrus species, these metabolites can be related with their different susceptibility/tolerance toward the Tristeza virus (CTV), one of the main biotic constraints for the citrus industry. The objective of this study was to compare the VOCs pattern from the leaves of a CTV-susceptible citrus variety such as Citrus aurantium and from three CTV-tolerant varieties: Citrus volkameriana, Carrizo citrange, and Forner-Alcaide no. 5. The VOCs emitted were analyzed via the headspace SPME method, while plant metabolites sequestered in the leaves were analyzed by heptane extraction followed by GC-MS. The results indicated that the majority of the VOCs emitted and sequestered in the leaves of the varieties tolerant and susceptible to CTV are constituted mainly by volatile terpenes (VTs) that exhibit strong qualitative/quantitative differences among the profiles of the four citrus species. In detail, the VOC emission indicated different patterns between C. aurantium and C. volkameriana and from both of them in comparison with Forner-Alcaide no. 5 and Carrizo citrange that exhibited more similarities, with the last two characterized by a higher presence of sesquiterpenes. The data obtained from the analysis of the VOCs sequestered in leaf tissues of the CTV-tolerant varieties indicated a higher presence of monoterpenes such as limonene, α-pinene, and p-cymene, known to be the main components of several plant extracts showing deterrent properties toward viruses and insect vectors. As VOC evaluation is a fast and noninvasive measure of phenotypic dynamics, allowing the association of plant phenotypes in accordance to plant disease resistance and/or stress tolerance, the possible implications of such differences in terms of tolerance grade to CTV and/or its related vectors are discussed.