Constitutive expression of the rice cold-inducible Osmyb4 gene in transgenic Arabidopsis (Arabidopsis thaliana) plants improves adaptive responses to cold and drought stress, most likely due to the constitutive activation of several stress-inducible pathways and to the accumulation of several compatible solutes (e.g., glucose, fructose, sucrose, proline, glycine betaine and some aromatic compounds). Although the Osmyb4 gene seems able to activate stress responsive pathways in different species, we previously reported that its specific effect on stress tolerance depends on the transformed species. In the present work, we report the effects of the Osmyb4 expression for improving the stress response in apple (Malus pumila Mill.) plants. Namely, we found that the ectopic expression of the Myb4 transcription factor improved physiological and biochemical adaptation to cold and drought stress and modified metabolite accumulation. Based on these results it may be of interest to use Osmyb4 as a tool for improving the productivity of woody perennials under environmental stress conditions.

Transgenic plants of grapefruit (Citrus paradisiMacf.) cvs. 'Duncan', 'Flame', 'Marsh', and 'Ruby Red' were obtained using Agrobacterium tumefaciens-mediated transformation of seedling epicotyl tissue. Two citrus tristeza virus (CTV)-derived candidate resistance genes: '392' (3′ region of the p23 ORF plus 3′ untranslated region-UTR) and 'p23 hairpin' (sense-p23 ORF plus UTR plus antisensep23 ORF) were introduced into grapefruit using Agrobacterium strains EHA105 and EHA101, respectively. Epicotyl explants from 1-mo.-old in vitro etiolated seedlings were incubated in bacterial suspension. Green shoots that formed on explants after 4-5 wk after bacterial incubation were tested for the presence of the GUS gene by histochemical analysis. The percentage of GUS-positive shoots and transformation efficiency was 30.3±3.3% and 3.5% for treatment with EHA101 and 15.3±1.7% and 1.3% for treatment with EHA105. GUS-positive shoots were micrografted onto Carrizo citrange (Citrus sinensis L. Osbeck×Poncirus trifoliata L. Raf.) seedling rootstocks, and the presence of transgene sequences in these plants was confirmed by polymerase chain reaction (PCR), Southern blot, and reverse transcription PCR analyses. Resulting transgenic grapefruit plants were challenged with CTV and tobacco mosaic virus using a protoplast challenge assay as an initial screen to determine the effects of the transgenes on virus replication. Although complete RNA-mediated resistance was not achieved, preliminary results showed that 5.2% of the recovered transgenic plants containing the '392' CTV-derived sequence repeatedly exhibited reduced CTV replication in protoplasts. These plants are being further evaluated using the traditional method of virus inoculation followed by enzyme-linked immunosorbent assay.

Somatic hybridization is a powerful tool for the genetic improvement of citrus rootstocks, and it is part of an efficient in vitro-based breeding system described here. An essential component of the system is the requirement of confirming tetraploidy and the combination of the two donor genomes. Expressed sequence tag–simple sequence repeat (EST-SSR) markers provide a means to accomplish both of these objectives, and their application to a population of pummelo [Citrus grandis (L.) Osbeck] + mandarin (C. reticulata Blanco) somatic hybrids developed for the specific purpose of providing alternative rootstocks for sour orange (Citrus aurantium L.) is detailed. Nineteen new somatic hybrids were produced from various mandarin and pummelo parents, and their ploidy level and the complementation of their nuclear genomes were confirmed using four EST-SSR markers. These markers were selected from markers previously mapped in sweet orange [C. sinensis (L.) Osbeck] and trifoliate orange [Poncirus trifoliata (L.) Raf.] and prescreened for suitable allelic polymorphism within the mandarin and pummelo lines used. After polymerase chain reaction amplification of sequences from the parents and putative hybrids, the products were separated on a genetic sequencer and visualized electronically. Additionally, EST-SSR markers identified the unexpected zygotic origin of a presumed nucellar embryogenic callus line. Integration of EST-SSR techniques for high-throughput genotyping with previously developed approaches to somatic hybrid creation increases substantially the effectiveness and efficiency of this in vitro-based breeding system for citrus rootstock improvement.

Transgenic grapefruit plants (Citrus paradisi cv. 'Duncan') with the isopentenyltransferase (ipt) gene under the control of APETALA3 promoter have been produced using Agrobacterium-mediated transformation. The relative expression level of the ipt gene was between 2.3 and 7 times higher in transformed plants than in the wild-type but despite the presence of a tissue-specific promoter, the expression was not limited only to flower tissue. Increased levels of trans-zeatin riboside between 9.4 and 32-fold found in transgenic grapefruit were considered the consequence of ectopic expression of the ipt gene. Chlorophyll levels in fully expanded uppermost leaves were also about 30% higher in transgenic than in wild-type plants. Involvement of cytokinins in control of expression of three pathogenesis-related protein genes: b-1,3-glucanase, a stress related PR gene 24P220, and an acidic chitinase, 24P262 was examined. Expression of b-1,3-glucanase, and 24P220 gene were significantly enhanced in transgenic plants while the expression of chitinase was reduced to low levels. Our results confirm the effect of cytokinins on expression of genes implicated in the response of grapefruit plants to pathogen attack and suggest a possible role of cytokinins in pathogen resistance.