Genomic and transcriptional analysis on the Mal d 1 allergen gene family in apple (Malus x domestica)

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sion of AhHMA4 may be useful in crops grown on soils poor in Zn. Acknowledgements This work was supported by FP6 EU PHIME project (FOOD-CT2006-016253). doi:10.1016/j.jbiotec.2010.09.767 [P-P&F.65] Moderate systemic movement of Cymbidium mosaic virus in Phalaenopsis seedling induced by Rhizoctonia orchid mycorrhizal fungi Ming-Chih Lee 1,∗ , Doris C.N. Chang 1 , Yu-Jie Chang 2 , Yu-Sen Chang 1 1

Department of Horticulture, National Taiwan University, Taiwan Graduate School of Environmental Education & Resources, Taipei Municipal University of Education, Taiwan Keywords: Phalaenopsis; Cymbidium mosaic virus (CymMV); Orchid mycorrhizal fungi (OMF); RT-PCR 2

Phalaenopsis orchid have gained worldwide popularity and grown extensively for international commercial flora in last decade. Susceptible to the viral disease, especially, caused by Cymbidium mosaic virus (CymMV), and free of disease-control has been a major concern among orchid growers due to their catastrophic effects on plant quality and market value. Hence this study tried to improve the resistance of Phalaenopsis to CymMV by root inoculation of beneficial and non-pathogenic orchid mycorrhizal fungi (OMF). The ex vitro micropropagated virus-free Phalaenopsis seedlings in four-leaf stage were symbiotic cultivated with three isolates of Rhizoctonia OMF (binucleate and multinucleate Rhizoctonia: BNR and MNR) for two months and subsequently inoculated with CymMV transcript. Reverse transcriptase polymerase chain reaction (RT-PCR) based detection of virus was conducted one week post virus challenge to confirm the movement of CymMV in systemic leaf of Phalaenopsis. Leaves of mycorrhizal Phalaenopsis Tai Lin Princess ‘V265 , which were with two months of symbiotic culture, challenged with CymMV showed a lower infection of CymMV than those of non-mycorrhizal controls. Similar plant responses were evenly obtained at the sixth month after virus challenged. Results from the current study indicated that mycorrhizal colonization of Phalaenopsis can effectively induced systemic viral resistance to CymMV via mechanism of restrictive systemic movement, whereas the protection efficacy were varied depending on the combination of OMF isolates and orchid cultivars. This study revealed that, systemic movement of CymMV in Phalaenopsis can be moderated by the mycorrhizal colonization of nonpathogenic BNR and MNR isolates. Concomitant increased in plant growth vigor and pathogenic resistance are suggested mechanism which induce systemic viral resistance of Phalaenopsis to the movement of CymMV. doi:10.1016/j.jbiotec.2010.09.768

[P-P&F.66] Specific detection of mycorrhizal colonization in orchid roots by fluorescence microscopy Ming-Chih Lee 1,∗ , Su-Feng Cheng 1 , Doris C.N. Chang 1 , Yu-Jie Chang 2 , Yu-Sen Chang 1 1

Department of Horticulture, National Taiwan University, Taiwan Graduate School of Environmental Education & Resources, Taipei Municipal University of Education, Taiwan Keywords: Orchid mycorrhizal fungi (OMF); Phalaenopsis; Anoectochilus formosanus Hayata; Fluorescent staining 2

Wild orchid roots always having fungi growing in their roots, this kind of orchid-fungus relationships are termed “orchid mycorrhiza” (OM). It plays nutritional roles in the early development of orchid seedlings and in whole life cycle of specific chlorophylldeficient species. Techniques to understand the nature of plant-fungal interactions and observe orchid mycorrhizal fungi (OMF) colonization in orchid roots are essential tools to mycorrhiza researchers. Nowadays, molecular tools and electron microscopy (EM) can be reliable tools but time-consuming, costly and high skill threshold. Routinely used traditional mycorrhizal observation are always presenting disadvantages of using toxic staining dye and provide relatively lower resolutions. Recently, a fluorescence microscopy based observation, which is time-saving, non-toxic and more specific, was developed. Orchid roots (Phalaenopsis sp. and Anoectochilus formosanus Hayata) with two month symbiotic cultivation were collected, washed and free-hand sectioned into thin layer and floated on water. For mycorrhizal control, root sections were soaked in 0.05% of aniline blue in lactoglycerin solution for three to four hours and then destained for use. The root sections then mounted in deion water on slides and observed under Olympus light microscope and fluorescence microscope with blue-light excitation (440 nm) and photographed by Nikon digital camera. Light-microscopy data indicated that aniline blue-staining and autofluorescence method are clearly indicated out the same OMF peletons within the infected cells. Under fluorescence microscope, OMFformed symbiosis structure in root sections of three orchids autofluoresced with green color under blue light excitation. Infection and peletons were greatly formed in cortex or entering into the endodermis or stele. Furthermore, individual hyphae of peloton could be easily viewed and differentiated from peleton-filled up root cells. In conclusion, this method is more specific than conventional aniline blue staining. doi:10.1016/j.jbiotec.2010.09.769 [P-P&F.67] Genomic and transcriptional analysis on the Mal d 1 allergen gene family in apple (Malus x domestica) Giulia Pagliarani 1,∗ , Roberta Paris 1 , Paul Arens 2 , Stefano 1 2 1 Tartarini , Eric van de Weg , Silviero Sansavini 1

Department of Fruit Tree and Woody Plant Sciences, University of Bologna, Italy 2 Plant Breeding, Wageningen-URl, Netherlands Keywords: Apple; Allergen; Mal d 1; PR-10 Apple (Malus x domestica) is one of the most economically important fruit species worlwide and it is considered also an important model species among woody perennial angiosperms due to the high quantity of genetic and genomic resources already available. Even if a regular apple consumpion is encouraged, it is one of the

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

fruits more frequently reported to cause allergic reactions. Among the four apple allergens know up to date, the main allergen Mal d 1 is classified as a complex gene family with at least 18 loci mainly clustered on Linkage groups (LG) 13 and 16. These genes encode for Pathogenesis Related proteins of class 10 (PR-10). In this work a genomic approach was firstly used to investigate the organization of Mal d 1 genes within the cluster on LG16. Through the sequencing of two Mal d 1 containing BAC clones much information was gained on genes orientation and distances, promoter sequences and on the relationship between the physical and genetic maps of this region. Interestingly, three new Mal d 1 genes were found in this cluster. Secondly, an highly specific tool was used to investigate the expression of Mal d 1 genes in apple fruit tissues and leaves. An expression partitioning was found for these genes among tissues, both at qualitative and quantitative levels and these results agree with the hypothesis of a subfunctionalization inside the Mal d 1 gene family, despite their high sequence and structural similarity. The availabilty of a comprehensive inventory of genes belonging to Mal d 1 family is a basal prerequisite to effectively study the contribution of individual Mal d 1 genes to allergenicity and the differential expression, retrieved in this study, may be an useful starting point to find out genes more involved in the determination of apple allergy. doi:10.1016/j.jbiotec.2010.09.770 [P-P&F.68] Setting up a Callogenesis and Transformation System for Globe Artichoke B. Menin 1 , S. Lanteri 1 , C. Comino 1 , A. Moglia 1,∗ , M. Barba 2 , T. Van Herpen 3 1

DIVAPRA Plant Genetics and Breeding, University of Torino, Italy CRA-PAV Plant Pathology Research Center, Rome, Italy 3 Plant Research International, P.O. Box 166700 AA Wageningen, Netherlands Keywords: Callus induction; Genetic transformation; Agrobacterium tumefaciens; Cynara cardunculus L. var. scolymus 2

Globe artichoke (Cynara cardunculus L. var. scolymus) is a species recalcitrant to genetic transformation, because of the difficulty of obtaining callus ‘in vitro’ and to regenerate a whole plant from it. We set up an efficient protocol to obtain callogenesis from leaf explants of virus-free plantlets obtained through meristem culture. A total of 108 combinations of light/dark exposure, hormone ratios, provision of polyphenol adsorbent compounds and polyphenol oxidase inhibitors were tested on two globe artichoke genotypes. The developed protocol made it possible to obtain a percentage of callogenesis close to 100% after only one week of culture. Callus induction was subsequently obtained from leaf explants after transformation mediated by Agrobacterium tumefaciens, containing the GUS marker gene. Regenerated calluses were subjected to histochemical analysis, and about 30% of them were positive to GUS assay. Globe artichoke, due to its natural exceptionally high content of caffeoylquinic acids (CQAs), represents a model species to study these compounds. We previously isolated and in vitro functionally characterized several enzymes (e.g. acyltransferases and hydroxylases) playing a key role in CQAs biosynthesis. The transformation protocol developed will be applied for investigating ‘in vivo’ the functional role of the isolated genes following their over-expression or down-regulation. A further step of our work will be the setting up of an effective organogene-

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sis protocol in order to regenerate globe artichoke transformed plants. doi:10.1016/j.jbiotec.2010.09.771 [P-P&F.69] TILLMore: a Forward- and a Reverse-Genetics Resource for the Identification of Root Morphology-Related Mutants R. Bovina 1,∗ , V. Talamè 1 , I. Szarejko 2 , G. Fincher 3 , R. Tuberosa 1 , M.C. Sanguineti 1 1

DiSTA, University of Bologna, Italy Department of Genetics, University of Silesia, Poland 3 ACPFG, Australia 2

In agriculture, water is usually the most limiting factor for plant growth. In this context, crops characterized by a more effective use of water will be at an advantage. Various morpho-physiological traits that have been shown to influence drought resistance but, among them, root morphology (shape, depth, size, number of root hairs, etc.) plays a crucial role. At DISTA, University of Bologna, a TILLING resource has been developed following chemical treatment with sodium azide (NaN3 ) on barley (cv. Morex). The resource, named TILLMore, can be considered for both forward- and reverse-genetics analyses (Talamè et al., 2008 Plant Biotechnol J 6: 477-485). The TILLING approach has been used to analyze four barley genes involved in root development (Brxl, Rpd1, HvExpˇ1and Miz1). This procedure allowed for the identification of 25 mutants corresponding to an average of six alleles per gene. Almost all the detected mutations were CG-to-TA transitions, as expected using sodium azide as mutagen agent, and several of them were missense, implying a change in amino acid sequence. In parallel, a forward-genetics analysis was performed on a portion of the mutagenized population to single out root morphology alterations by comparison with Morex wild-type. For this purpose a simple paper-roll approach was performed allowing for the identification of phenotypic variants at the seedling stage. The analysis was completed on ca. 1,000 M3 families and the phenotypic evaluation was repeated only for the putative mutants identified during the preliminary screening. The screening for root phenotype at the seedling stage allowed us to identify a total number of ca. 70 lines with altered root morphology, corresponding to ca. 7% of the families. A more accurate phenotypic characterization of the mutant lines is currently in progress. doi:10.1016/j.jbiotec.2010.09.772 [P-P&F.70] Starch Metabolism Mutants in Barley: a TILLING Approach R. Bovina 1,∗ , V. Talamè 1 , P. Trost 2 , F. Sparla 2 , M.C. Sanguineti 1 , R. Tuberosa 1 1

DiSTA, University of Bologna, Italy Department of Experimental Evolutionary Biology, University of Bologna, Italy 2

At DiSTA (University of Bologna), a sodium azide-mutagenized barley (cv. Morex) population of ca. 5,000 M3 families, named TILLMore, has been developed for identifying mutants at target genes using the TILLING procedure (McCallum et al., 2000). Until now the TILLMore resource has been screened for almost 20 genes thus resulting in a frequency of mutation of 1 mutation every 430 Kb. We utilized the TILLING approach to identify mutants