Literatura académica sobre el tema ""grapevine transformation""

A major drawback to the use of embryogenic cultures for transformation of grapevine is that their ability to undergo genetic transformation is cultivar-dependent. Also, depending on cultivar, embryogenic cultures are difficult to impossible to maintain over time, reducing their utility for use in genetic transformation. An alternative to the use of embryogenic cultures for transformation of grapevine is the use of micropropagation cultures, which are easier to initiate from a wide range of grapevine cultivars and can be maintained over time without loss of function. Vitis vinifera `Thompson Seedless' was used as a model for genetic transformation using micropropagation cultures. In vitro cultures were initiated from apical meristems of actively growing vines and maintained in C2D medium containing 4 μM of 6-benzylaminopurine (C2D4B). Shoot tips and nodes were collected from proliferating in vitro cultures for transformation studies. A variety of wounding techniques, including nicking, sonication, and fragmenting of meristematic tissues was employed in order to enable Agrobacterium infection. We used a construct containing a bidirectional 35S promoter complex with a marker gene composed of a bifunctional fusion between an enhanced green fluorescent protein (EGFP) gene and a neomycin phosphotransferase (NPTII) gene in one direction and a hybrid lytic peptide gene in the other. Transgenic shoots growing in C2D4B medium containing 200 mg·L-1 each of carbenicillin and cefotaxime and 20 mg·L-1 of kanamycin were selected based on GFP fluorescence. Transgenic shoots were rooted and transferred to a greenhouse. To date, 18 transgenic lines have been generated. Details on the transformation procedure will be discussed.

In the 90s, as in other countries, transformation of Chilean viticulture brought about the introduction and spread of European grapevine varieties which has resulted in a massive loss of minor local and autochthonous grapevine varieties traditionally grown in several wine growing regions. Fortunately, in recent years, autochthonous and minority varieties have been revalued due to their high tolerance to pests and diseases and because of their adaptation to thermal and water stress triggered by global warming. In this study, we assessed the nutritional status of two autochthonous grapevines grafted onto four different rootstocks under the hyper-arid climatic conditions of Northern Chile over three consecutive seasons. The results showed that R32 rootstock induced high N, P, Ca, Mg and Mn levels in blades compared to Harmony rootstock. R32 rootstock and to a lesser extent, 1103 Paulsen and 140 Ruggeri rootstocks kept balanced levels of nutrients in blades collected from Moscatel Amarilla and Moscatel Negra grapevine varieties. Additionally, Harmony presented slight nutritional imbalance compared to the rest of studied rootstocks due to its low absorption of Mg, Mn, Ca and P, and its high K absorption, which was exacerbated under warm weather and salinity soil conditions. These results may provide a basis for specific cultivar/rootstock/site combinations, a nutritional guide for the viticulturists of Northern Chile, and options to diversify their production favoring the use of minority and autochthonous varieties that adapt well to hyper-arid conditions of Northern Chile.

AbstractThe grapevine (Vitis vinifera) genome was analyzed in silico for homologues of plant genes involved in Agrobacterium transformation in Arabidopsis thaliana and Nicotiana spp. Grapevine homologues of the glucomannan 4-betamannosyltransferase 9 gene CslA-09 involved in bacterial attachment to the cell wall, homologues of reticulon-like proteins BTI1, 2, 3 and RAB8 GTPases, both involved in T-DNA transfer to the host cell, homologues of VirE2 interacting protein VIP1 that contributes to the targeting of T-DNA into the nucleus and to its integration, and homologues of the histone protein H2A, which promotes the expression of T-DNA encoded genes, were selected. Sequences homologous to the arabinogalactan-protein AtAGP17 were not found in the grape genome. Seventeen selected candidates were tested by semiquantitative RT-PCR analysis for changes in their expression levels upon inoculation with Agrobacterium tumefaciens C58. Of the tested homologues, the expression of VvRab8a, VvVip1a and two histone genes (VvHta2 and VvHta10) increased significantly, therefore we hypothesize that these might be involved in Agrobacterium transformation of V. vinifera.

Thesis (MSc)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Agrobacterium tumefaciens-mediated transformation technology has been used in a variety of applications throughout the fields of cellular and molecular plant biology as well as plant physiology. Research is conducted in order to extend this application range and overcome some of the intrinsic limitations of the Agrobacterium transformation system. Predominantly, these limitations can be attributed to the host range specificity of A. tumefaciens, as well as adverse effects induced on explant tissue by active plant defence mechanisms, triggered by the plant-pathogen-interaction. Typically, this active defence mechanism culminates in the hypersensitive response (HR), characterised by localised cell death and necrosis. Not all Agrobacterium species, however, share the same host range and some have evolved the ability to infect plant species not normally considered hosts of A. tumefaciens. This host range specificity can be exploited to extend the application of existing Agrobacterium transformation systems. In an attempt to establish an efficient transformation system for Vitis vinifera which, has proven very difficult to transform with A. tumefaciens, indigenous A. vitis strains have been evaluated as possible host-specific transformation agents. Strains of Agrobacterium vitis should be suitable for this type of endeavour, since they have evolved several unique characteristics directly linked to the infection of their hosts. These include the ability to utilise, tartrate, a host abundant carbon source, as well as the production of an acid polygalacturonase that could play a role during the infection process. The proposition that the evolution of A. vitis is a fairly recent event is also confirmed by the relatively little divergence observed between A. tumefaciens and A. vitis. In this study, a selection of A. vitis strains were evaluated in screenings designed to accentuate desirable traits in strains such as good infectivity of grapevine material (presumably an indicator of an efficient mechanism of gene transfer to be exploited in an engineered transformation system) as well as a favourable reaction (causing no necrosis) on grapevine somatic embryos. Two strains produced large tumours on grapevine cuttings and caused little necrosis on the somatic embryos. Significant variation in infectivity as well as callus necrosis was observed between the strains as well as in a genotype-specific manner on the host material. This genotypic-specific effect of either host or pathogen could be an indication of the degree of specialisation developed by plant pathogens to infect specific hosts. On the basis of these results, it was possible to select an A. vitis strain for further biochemical and genetic characterisation. Simple biochemical analysis classified the strain as an octopine strain. DNA-DNA hybridisation techniques combined with a plasmid walking technique resulted in the partial characterisation of the T-DNA of the selected A. vitis strain. A partial restriction enzyme map of the T-DNA was constructed and the T-DNA and flanking areas were cloned. Significant differences, most notably, the absence of a TB-area as well as the absence of the agrocinopine (aes) gene from the 5' area of the T-DNA, were observed. Partial sequencing data indicated the presence of at least four conserved T-DNA genes located on the TA-DNA, as well as the presence of three bacterial insertion (IS-)elements flanking the region. Two of these IS elements, both related to the IS 110 family of IS elements have not yet been reported in A. vitis. In fact, these two elements seem to be the 5' and 3' ends of a disrupted element and could therefore have played an evolutionary role in the development of this strain. This study provides fundamental background for the development of a more efficient transformation system specific for grapevine, exploiting same of-the unique characteristics of one of its pathogens, A. vitis.
AFRIKAANSE OPSOMMING: Agrobacterium tumefaciens-gebaseerde transformasiesisteme word in "n wye reeks van toepassings in die velde van sellulêre- en molekulêre plantbiologie asook plantfisiologie aangewend. Navorsing word voortdurend onderneem om die inherente beperkinge van die Agrobacterium-transformasiesisteem te oorkom en sodoende die toepassingsveld van die sisteem verder te verbreed. Die beperkinge tipies aan dié sisteem kan hoofsaaklik toegeskryf word aan die gasheerspesifisteit van A. tumeteciens, asook die negatiewe reaksies op eksplantmateriaal wat deur die plant se aktiewe verdedigingsmeganisme, soos ontlok deur die plant-patogeen interaksie, veroorsaak word. Hierdie aktiewe verdedigingsmeganisme lei gewoonlik tot In hipersensitiewe respons (HR) in die plant, wat deur gelokaliseerde selafsterwing en nekrose gekenmerk word. Alle Agrobacterium-spesies het egter nie almal dieselfde gasheerreeks nie en sommige rasse het as gevolg van evolusionêre ontwikkelings die vermoë verkry om plantspesies wat normaalweg buite die gasheerreeks van A. tumefaciens val, te infekteer. Hierdie tipe gasheerspesifisiteit kan uitgebuit word om die toepassingsmoontlikhede van bestaande Agrobacterium-transformasiesisteme te verbreed. In In poging om In effektiewe transformasiesisteem vir Vitis vinifera, In moeilik transformeerbare gewas, te ontwikkel, is inheemse rasse van Agrobacterium vitis ondersoek as moontlike gasheerspesifieke transformasie-agente. Rasse van A. vitis behoort uiters geskik te wees vir so "n toepassing, aangesien hulle verskeie unieke eienskappe, wat direk aan die infeksie van die gasheer gekoppel is, vertoon. Van hierdie eienskappe is onder meer die vermoë om tartraat, In koolstofbron volop in druifplante, te benut. A. vitis produseer verder ook In suur poligalaktorunase wat vermoedelik In rol in die infeksieproses speel. Die voorstel dat die evolusionêre ontwikkeling van A. vitis In redelike onlangse gebeurtenis is, word onderskryf deur die betreklike homogenisiteit met A. tumefaciens. In hierdie studie is "n groep A. vitis-rasse met behulp van siftingsprosedures wat daarop gemik is om gesogte eienskappe in rasse uit te wys, beoordeel. Die vermoë om druifplantmateriaal te infekteer (wat vermoedelik "n aanwyser van "n effektiewe meganisme van geenoordraging is wat in "n gemanipuleerde transformasiesisteem benut kan word), sowel as 'n gunstige reaksie (d.w.s geen nekrose) op druifplant somatiese embrio's is van die gesogte eienskappe waarvoor gesoek word. Twee rasse het groot tumors op druifplant-stingelsegmente veroorsaak terwyl hulle bykans geen weefselskade op somatiese embrio's geïnduseer het nie. Betekenisvolle verskille in infektiwiteit en in kallusnekrose is tussen die rasse sowel as in 'n genotipe-spesifieke-verhouding waargeneem. Hierdie genotipe-spesifieke effek, kenmerkend van óf die gasheer óf die patogeen, kan aanduidend wees van die vlak van spesialisasie wat heers by die infeksie van spesifieke gashere. Na aanleiding van bogenoemde resultate was dit moontlik om 'n A. vitis-ras te selekteer wat verder aan biochemiese en genetiese analises onderwerp kon word. Eenvoudige biochemiese analises het dit moontlik gemaak om die ras as oktopien te klassifiseer. DNA-DNA hibridisasietegnieke gekombineerd met 'n unieke plasmiedwandeltegniek het gelei tot die gedeeltelike karakterisering van die geselekteerde A. vitisras. In Gedeeltelike restriksie-ensiem (RE) kaart van die T-DNA kon gevolglik opgestel word. Die T-DNA en die aangrensende gedeeltes is boonop gekloneer. Betekenisvolle verskille, spesifiek die afwesigheid van In TB area, sowel as die afwesigheid van die agrosinopien-sintasegeen (acs) aan die 51-kant van die T-DNA, is waargeneem. Gedeeltelike basispaaropeenvolgingsdata het egter die teenwoordigheid van minstens vier gekonserveerde T-DNA-gene, asook die teenwoordigheid van drie bakteriese invoegingselemente (IS) aan weerskante van die area, geïdentifiseer. Twee van hierdie elemente, wat beide homologie vertoon met die IS110 familie van IS elemente, is nog nie vantevore in A. vitis aangetref nie. Dit wil boonop blyk of dié twee elemente die 51 - en 31 - areas van In onderbroke element vorm, wat dus In moontlike aanduiding is van hul potensiële rol in die evolusionêre ontwikkeling van die ras. Hierdie studie verskaf basiese inligting wat daartoe kan lei dat 'n doeltreffender transformasiesisteem spesifiek vir druifplante ontwikkel word deur van die unieke kenmerke van een van sy patogene, A. vitis, uit te buit.

Doutoramento em Engenharia Agronómica - Instituto Superior de Agronomia
Considering the growing importance of black foot disease of grapevine, this study was aimed to deeply understand details on taxonomy, genetics, biology and pathological behaviour of its main causal agents, previously attributed mostly to Ilyonectria liriodendri and I. macrodidyma. A multi-gene analysis of a collection of Ilyonectria isolates, along with morphological characterisation, enabled the description of 12 species from I. radicicola and four from I. macrodidyma complexes. Among these, pathogenicity experiments revealed I. lusitanica, I. estremocensis and I. europaea as more virulent to grapevine than I. liriodendri and I. macrodidyma. The entire mating-type loci of I. liriodendri and of species from the I. macrodidyma complex were obtained. While the idiomorph structure of species from the latter matches that of other heterothallic Hypocreales, the organization of the mating-type loci in I. liriodendri seems unique, suggesting a potential pseudo-heterothallism. Soilborne inoculum is accepted to contribute significantly to initiate black foot disease in grapevine plants. qPCR amplification from DNA soil samples demonstrate that rotation can reduce the levels of Ilyonectria in nurseries, and that levels of infestation in vineyard soils are lower than in nursery or mother-plant soils. Additionally, a protoplast transformation protocol is presented for the stable integration of the GFP gene in the genome of I. liriondendri, enabling future downstream functional genetic studies.

Dans cette étude, nous avons dans un premier temps adapte le protocole d'embryogenèse somatique primaire a différentes variétés d'hybrides porte-greffes (3309C, 110R, Fercal, 41B et SO4) en nous appuyant sur l'expérience acquise au laboratoire sur Vitis vinifera cv Chardonnay. Les résultats montrent que le génotype, le type d'explant (étamine, fleur ou nœud), le type et la dose d'auxine utilisés dans le milieu d’induction (2,4-D ou 2,4,5-T) ont une influence sur les efficacités d'embryogenèse somatique. En effet, pour le 3309C, l'utilisation du 2,4,5-T dans le milieu d'induction a montré une efficacité embryogène supérieure à partir de nœuds par rapport à celle obtenue à partir d'étamines. Cependant la meilleure efficacité a été obtenue à partir de fleurs de cette variété, sur un milieu d'induction contenant du 2,4-D. De plus, le protocole d'embryogenèse somatique secondaire utilise de manière récurrente au laboratoire nous a permis d'obtenir des masses embryogènes ainsi que des embryons somatiques secondaires de ces porte-greffes. Le protocole de conversion des embryons en plantes, en présence de 4,5 uM de cytokinine (BAP) s'est avère efficace pour le 11OR et le 41B. Dans un second temps, nous avons co-cultivé le matériel embryogène obtenu pour quatre de ces génotypes (110R, 3309C, Fercal et 41B), avec Agrobacterium tumefaciens contenant trois constructions génétiques : (i) une copie d'une séquence partielle (1020 pb) du gène de la coque protéique du virus en orientation sens; (ii) une partie courte en sens et en anti-sens (280 pb) de cette même séquence formant une structure en épingle a cheveux (hpRNA = hairpin RNA) ; (iii) un amiRNA ciblant une séquence virale. Le gène bactérien codant la néomycine phosphotransférase et conférant la résistance à un antibiotique, la kanamycine, a été utilisé comme gène de sélection. Les conditions de sélection a la kanamycine ont nécessité des adaptations expérimentales telles que l’ajustement de la concentration en antibiotique puisque la sélection avec 75 mg.L-1 de kanamycine s'avère insuffisamment drastique dans Ia plupart de nos expériences de co-cullture. Les résultats d'analyse moléculaire par PCR ont montré l'amplification probable des fragments d'intérêt (CPGFLV et amiRI1TA-71) dans des échantillons de 11OR et de 41B résistants à la kanamycine. Cependant des analyses moléculaires supplémentaires par AL-PCR ne nous ont pas renseignées sur une éventuelle intégration du transgène amiRATA-71 dans des masses embryogènes de 41B
In this study, we initially adapted the protocol of primary somatic embryogenesis in different varieties of hybrid rootstocks (3309C, 110R, Fercal, 41B and SO4) building on the experience gained in the laboratory on Vitis vinifera cv Chardonnay. The results show that the genotype, the explant type (stamen, flower or node), the type and the dose of auxin used in the induction medium (2,4-D or 2,4,5-T) influence the efficiency of somatic embryogenesis. Indeed, for the 3309C, the use of 2,4,5-T in the induction medium showed a higher efficiency from embryogenic nodes compared to that obtained from stamens. However, the better efficiency was obtained from the flowers of this variety on an induction medium containing 2,4-D. In addition, a protocol used in the laboratory for secondary somatic embryogenesis allowed us to obtain embryogenic masses as well as secondary somatic embryos from these rootstocks. The protocol conversion of embryos into plants, in the presence of 4.5 [tM of cytokinin (BAP), was effective for the 110R and 41B. In a second step, we co-cultivated embryogenic material obtained for four of these genotypes (110R, 3309C, Fercal and 41B), with Agrobacteriwn tumefaciens containing three genetic constructs: (i) a copy of a partial sequence (1020 bp) of the coat protein gene of the virus in the sense orientation, (ii) a short part-way and antisense (280 bp) of the same sequence forming a hairpin structure (hairpin RNA = hpRNA) (iii) one amiRNA targeting a viral sequence. The nptll bacterial gene encoding neomycin phosphotransferase and conferring resistance to the antibiotic kanamycin, was used as the selection gene. The selection conditions to kanamycin have required experimental adaptations such as adjusting the concentration of antibiotic because the selection with 75 mg.L-1 of kanamycin was not enough drastic in most of our experiments of co-culture. The results of molecular analysis by PCR showed probable amplification of fragments of interest (CPGFLV and amiRNA-71) in samples of 11OR and 41B resistant to kanamycin. However, additional molecular analysis by AL-PCR did not inform us about a possible integration of the transgene amiRNA-71 in embryogenic masses of 41B

Les objectifs de ce travail etaient de developper des methodologies d'induction de l'embryogenese somatique et de transformation genetique en vue d'obtenir des vignes transgeniques susceptibles d'etre resistantes au gravepine fanleaf virus (gflv). Un protocole d'embryogenese somatique a partir de culture d'etamines a ete developpe. Ce protocole a permis l'obtention de cals embryogenes de cepages d'interet tels que les vitis vinifera cv. Riesling et cv. Pinot noir. Nous avons egalement developpe un protocole d'embryogenese somatique chez le porte-greffe fercal base sur la mise en culture de fragments de feuilles provenant de vitroplants. Nous avons, par ailleurs, realise une etude histologique de l'embryogenese somatique qui a permis de mettre en evidence l'origine des cals embryogenes. Les cals embryogenes de fercal proviennent de la proliferation de cellules du mesophylle situees autour des nervures. Nous avons confirme l'origine unicellulaire des embryons en suivant le developpement de ceux-ci depuis la premiere division asymetrique conduisant a l'elaboration de l'embryon et de son suspenseur a sa base. Les caracteristiques morphologiques et cytologiques des cals embryogenes issus de la culture d'etamines ont ete determinees. Les cals embryogenes sont constitues de deux lignees cellulaires : l'une avec des amyloplastes volumineux et l'autre qui en est depourvue. Nous avons montre que l'amidon et la callose sont des marqueurs de l'embryogenese somatique chez la vigne. Enfin une partie de ce memoire retrace nos experiences de transformation genetique des cals embryogenes de 110r par agrobacterium tumefaciens en vue d'introduire une resistance au gflv. Des vignes transgeniques ont ete produites avec quatre constructions plasmidiques differentes. Nous avons transfere une copie du gene codant pour la proteine de capside du gflv, une version mutee du gene codant pour l'arn polymerase du gflv, ou une sequence codant pour le polypeptyde vpg-protease. Les transformants regeneres ont ete analyses par pcr et southern blot pour confirmer le transfert et l'insertion des t-dna. Un test de microgreffage a ete developpe pour mettre en evidence une resistance a l'infection par le gflv chez les vignes transgeniques. Deux transformants ppo1-r (a2 et a10) ont ete identifies comme etant resistants au gflv par microgreffage in vitro. Des microgreffes de ces deux transformants ne presentent pas d'infection au gflv apres 4 mois d'assemblage entre les greffons transgeniques et l'inoculum viral, au contraire des temoins non transgeniques.

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies.
AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.

La vite è una delle più importanti piante da frutto coltivate nel mondo ed il suo valore economico è per la maggior parte dovuto alla produzione vitivinicola. La totalità delle piante coltivate sul territorio Europeo appartiene alla specie Vitis vinifera L., una pianta in grado di produrre uva di elevata qualità ma purtroppo è suscettibile a varie malattie, che causano rilevanti perdite di raccolto in tutto il mondo. Nonostante la disponibilità di prodotti fitosanitari in grado di controllare la maggior parte delle patologie della vite, le problematiche agro-ecologiche legate all’utilizzo di prodotti chimici in agricoltura hanno incoraggiato la comunità scientifica a ricercare metodi alternativi per il controllo dei patogeni in campo, cercando di ridurre le influenze sia economiche che ecologiche dei fitofarmaci sulla viticoltura. Nello specifico è noto che alcune specie selvatiche di vite, originarie del Nord America e dell’Asia, posseggono vari livelli di resistenza nei confronti di diversi patogeni. Nonostante queste fonti naturali di resistenza siano state utilizzate in passato per la produzione di ibridi interspecifici resistenti, nessuno di questi è stato totalmente accettato dai produttori vitivinicoli a causa dei numerosi difetti organolettici legati alla persistenza delle caratteristiche tipiche delle viti selvatiche. Un significativo passo in avanti è stato compiuto nel 2007, anno in cui è stato completa il sequenziamento del genoma di Vitis vinifera L. Da allora, una più profonda conoscenza delle ragioni genetiche alla base delle caratteristiche delle bacche e della resistenza ai patogeni ha aiutato la comunità scientifica ad affrontare il problema della suscettibilità della vite ai patogeni mediante prevalentemente due approcci: la selezione assistita da marcatori (MAS) e la trasformazione genetica. Questo progetto di dottorato si è focalizzato sul secondo approccio, nello specifico siamo riusciti a produrre viti transgeniche caratterizzate da una maggiore resistenza nei confronti di Plasmopara viticola, l’agente eziologico della peronospora della vite. In precedenza il nostro gruppo di ricerca si era occupato dello studio delle differenze trascrittomiche in risposta all’infezione da P. viticola nella vite suscettibile (V. vinifera) ed in una naturalmente resistente (V. riparia). Otto geni, specificamente indotti solamente nella vite resistente in seguito all’infezione, sono stati identificati come simili ad ATLs (Arabidopsis toxicos en Levadura), una famiglia genica nota in altre specie per essere indotta da elicitori. VrATL2, un ortologo di Arabidopsis thaliana ATL2, è stato selezionato come promettente gene candidato per la trasformazione di vite. Dato che la famiglia genica ATL era sostanzialmente sconosciuta in vite, il primo passo è stato quello di definirne i membri. Utilizzando il caratteristico dominio RING-H2 come sonda per un’analisi PSI-Blast all’interno del genoma tradotto di V. vinifera cv. Pinot Noir abbiamo identificato tutti i trascritti appartenenti a questa famiglia genica in vite. La famiglia genica è quindi stata analizzata definendo le caratteristiche molecolari di ciascun membro, le relazioni filogenetiche e i profili di espressione genica in differenti tessuti e stadi di sviluppo di vite. Successivamente sono state messe a punto le tecniche per l’induzione ed il mantenimento di calli embriogenetici di vite e quindi abbiamo ottenuto la trasformazione stabile di piante di V. vinifera cv. Shiraz con un’espressione aumentata e costitutiva del gene VrATL2. Le piante rigenerate sono quindi state caratterizzate da un punto di vista molecolare mediante analisi di Southern blot e Real Time qPCR, in modo da definire precisamente il numero di inserzioni e l’effettivo livello di espressione del transgene. Siamo quindi passati alla fenotipizzazione delle piante transgeniche rispetto alla resistenza nei confronti di P. viticola mediante due differenti metodi: l’osservazione microscopica dell’area di sporulazione e il calcolo della stessa mediante un software di elaborazione di immagini. Con lo scopo di descrivere ulteriormente il fenotipo osservato, tre linee rappresentative sono state selezionate per studiare le differenze trascrittomiche, dovute alla presenza del transgene, mediante un esperimento di microarray. Infine nelle stesse piante è stato anche studiato il processo di infezione mediante l’osservazione microscopica dello stesso a differenti intervalli temporali. L’ultima parte di questo progetto di dottorato è stata dedicata allo studio delle regioni regolatrici del gene ATL2 in entrambe le specie di vite. Le due regioni sono state isolate del DNA genomico di V. vinifera e V. riparia e successivamente analizzate mediante software bioinformatici per predire la struttura dei promotori e la presenza di specifici elementi regolatori. Quindi l’effettiva capacità di promuovere la trascrizione di un gene reporter è stata verificata mediante l’analisi funzionale in piante modello: trasformazione transiente di Nicotiana Benthamiana e trasformazione stabile di Arabidopsis thaliana. Infine abbiamo curato la messa a punto di un database interrogabile di tutti gli elementi regolatori predetti nei promotori dei geni ATL in vite; questo database potrà in futuro fornire spunti utili per indirizzare ulteriori analisi funzionali di questa famiglia genica. In conclusione, i risultati ottenuti durante questo dottorato di ricerca hanno dimostrato che nonostante la trasformazione stabile di vite sia ancora una metodica lunga e laboriosa, tanto da scoraggiare esperimenti di analisi funzionale, l’accurata scelta di un gene candidato può portare a buoni risultati in termini di fenotipo atteso. Inoltre, ad oggi la trasformazione stabile resta sostanzialmente il metodo di eccellenza per le analisi funzionali in vite. In linea con quanto detto siamo riusciti a generare viti transgeniche caratterizzate dall’aumentata resistenza noi confronti di P. viticola mediante la sovra espressione costitutiva del gene ATL2 isolato da V. riparia. Nonostante siano necessari ulteriori esperimenti per confermare la durata della resistenza osservata e per valutare accuratamente il fenotipo delle piante trasformate, speriamo che in futuro questo approccio possa contribuire a sviluppare una difesa antiperonosporica più sostenibile.
Grapevine is one of the most important fruit crop in the world, with a high economic impact mainly due to the wine production. The European Vitis vinifera L. produces high quality grapes but is prone to several pathogens, which cause significant losses to viticulture worldwide. Even if chemical control is available for most of the diseases, agro-ecological concerns pushed the scientific community to search for alternative methods, in order to reduce both the environmental and economic issues associated to pest control. In particular several wild species of the genus Vitis from North America and Eastern Asia exhibit various levels of natural resistance towards distinct pathogens. Despite these natural resistance sources have been used to in the past to produce first resistant hybrids, none of them reached complete growers acceptance because of adverse organoleptic features. A significant step forward was achieved in the 2007, when the Vitis vinifera L. genome was completely sequenced. Since then the deeper knowledge of genetic determinants of both resistance and berry quality traits boosted the grapes scientific community in facing the Vitis susceptibility mainly by two different approaches: marker associated selection (MAS) breeding and genetic transformation. This PhD project was focused on the second approach, in particular we carried out the stable transformation of grapevine in order to enhance its resistance towards Plasmopara viticola, the causative agent of downy mildew. Transcriptomic responses to P. viticola in both the susceptible V. vinifera and the resistant V. riparia have been previously studied by our research group. Eight genes, specifically induced only in V. riparia upon the infection process, were identified as similar to ATLs (Arabidopsis Toxicos en Levadura), a gene family known to be rapidly induced by common elicitors. VrATL2, an ortholog of Arabidopsis thaliana ATL2, was selected as a promising candidate gene for stable grapevine transformation. As the ALT gene family was almost unknown in grapevine, the first step was the complete survey of the family members. The canonical RING-H2 domain was used as bite to search for putative ATLs within the translated genome of V. vinifera cv. Pinot Noir by PSI-Blast analysis. The resulting protein family was manually curated and analysed for specific molecular characteristics, phylogenesis and gene expression profiles in different grapevine tissues and developmental stages. Once set up the induction and maintenance of grapevine embryogenic material, we stably transformed V. vinifera cv. Shiraz producing plants with increased constitutive expression of VrATL2. The newly generated plants were molecularly characterised by Southern blot and Real Time qPCR analyses in terms of number of insertions and actual level of transgene expression respectively. The phenotyping of transformed plants for their resistance against P. viticola was carried out by means of two different methods: microscopical visual inspection and computational image analysis. The observed phenotype was further described analysing the transcriptomic changes in three selected transgenic lines by a microarray experiment and finally, on the same lines, the infection process was evaluated by microscopic observations of a time-course experiment. The last part of this PhD project was focused on the characterization of the ALT2 regulative regions from V. vinifera and V. riparia. After the bioinformatic analysis of the isolated regulative regions in terms of promoter structure and cis-acting elements, their ability to promote the transcription in heterologous systems was verified, by transient transformation of Nicotiana benthamiana and stable transformation of Arabidopsis thaliana. Lastly, we implemented the setup of a custom interrogable database of all cis-acting elements predicted for the ATL gene family in grapevine, which might facilitate further analysis within this family. In conclusion, this PhD project showed that even if stable transformation of grapevine remains an arduous and time-consuming task, and functional analysis are almost precluded, the accurate and informed choice of a candidate gene may provide good results in terms of expected phenotype. Moreover, the actual stable transgenic expression remains the most informative and plausible approach for functional analysis in V. vinifera plants. Indeed we were able to produce transgenic grapevines with enhanced resistance towards P. viticola by constitutive overexpression of the ATL2 gene. Despite further experiments are needed to confirm the durability of the observed resistance and to describe the associated phenotypic features over the time, we hope that in the future this approach could help in terms of sustainable agriculture and food safety.

La vite (Vitis vinifera L.), una delle più coltivate piante da frutto, riveste notevole importanza economica in tutto il mondo. Poichè negli ultimi decenni la viticoltura sta subendo gli effetti del riscaldamento globale (Webb et al., 2007), è necessario mantenere una produzione di uva e vino di elevata qualità. Una delle maggiori sfide consiste nell’identificazione dei principali geni regolatori dello sviluppo della pianta di vite durante il ciclo vegetale annuale e, in particolare, della transizione dalla fase vegetativa a quella matura (detta véraison), durante la quale avvengono profonde modificazioni biochimiche, fisiologiche e trascrizionali. Grazie ad un'analisi di network di co-espressione sull’atlante del trascrittoma della vite e ad un dataset di dati trascrizionali di bacche (Massonnet, 2015; Palumbo et al., 2014; Fasoli et al., 2012), è stata identificata una nuova categoria di geni chiamata 'switch’; tali geni sono significativamente up-regolati durante la transizione di fase ed inversamente correlati a molti geni soppressi durante la fase matura. Tra questi, i fattori di trascrizione NAM/ATAF/CUC (NAC) rappresentano un’interessante famiglia genica dato il ruolo chiave in processi biologici come sviluppo e risposte allo stress in pianta (Jensen et al., 2014). Per la caratterizzazione funzionale cinque geni NAC sono stati selezionati come putativi principali regolatori della riprogrammazione del trascrittoma durante la maturazione della vite. VvNAC11, VvNAC13, VvNAC33 e VvNAC60 sono stati identificati come geni 'switch' dalla sopra citata analisi, mentre VvNAC03 come gene omologo a NOR (non-ripening) di pomodoro, uno dei principali regolatori della maturazione di tale frutto (Giovannoni, 2004; Giovannoni et al., 1995). I cinque NAC sono stati sovra-espressi transientemente in Vitis vinifera per ottenere una panoramica dei loro effetti primari sul trascrittoma. Sono poi state ottenute e caratterizzate dal punto di vista molecolare e fenotipico piante di vite stabilmente trasformate con VvNAC33 e VvNAC60. VvNAC33 sembra essere coinvolto nella regolazione negativa della fotosintesi poiché le foglie sovra-esprimenti tale gene contengono una minor quantità di clorofilla, mentre VvNAC60 provoca una ridotta crescita della pianta e una prematura lignificazione dello stelo rispetto ad una pianta controllo della stessa età. Questi risultati riflettono comportamenti tipici di piante in fase di maturazione e senescenza, sostenendo l’ipotesi di un ruolo fondamentale dei NAC nella transizione di fase in vite. Al fine di identificare i target che agiscono a valle dei NAC, sono state eseguite analisi microarray sulle foglie delle piante trasformate in modo transiente e stabile. In entrambe le over-espressioni è stata influenzata l’espressione di un'ampia gamma di processi cellulari tra cui, tra le categorie funzionali più rappresentate, vi sono trasporto, metabolismo secondario e attività dei fattori di trascrizione. L'identificazione di VvMYBA1, un noto regolatore della biosintesi degli anotciani in vite (Kobayashi et al., 2002), come target di VvNAC60 suggerisce un ruolo di tale NAC in processi tipici dell’inizio della maturazione. Un altro approccio utilizzato in questo lavoro è stato la complementazione funzionale del mutante nor di pomodoro con i NAC selezionati. Risultati preliminari hanno rivelato che VvNAC03 e VvNAC60 sembrano avere una funzione simile a NOR poichè riescono a maturare almeno esternamente. In conclusione, i risultati ottenuti in questo lavoro suggeriscono la capacità dei VvNAC selezionati di influenzare l'espressione di geni coinvolti nella regolazione che controlla lo sviluppo dalla fase vegeativa alla fase matura in vite. Questo lavoro ha inizato a far luce sul ruolo dei NAC nello sviluppo della vite, ma dovranno essere effettuate ulteriori analisi per ottenere una piena compresione del macchinario molecolare che regola questo complesso sistema di regolazione.
Grapevine is the most widely cultivated and economically important fruit crop in the world. Viticulture has been affected by the global warming currently under way over the past few decades (Webb et al., 2007). Improving the genetics of key grapevine functions is needed to keep producing high quality grapes and wine. In this context, a challenging task is to identify master regulators that program the development of grapevine organs and control transition from vegetative-to-mature growth featured by grape berries during the annual plant cycle. This transition, called véraison, is marked by profound biochemical, physiological and transcriptomic modifications that allow vegetative green berries to enter the ripening process. Thanks to an integrated network analysis performed on the grapevine global gene expression atlas and from a large berry transcriptomic data set (Massonnet, 2015; Palumbo et al., 2014; Fasoli et al., 2012) a new category of genes, called ‘switch’ genes, was identified; they were significantly up-regulated during the developmental shift and inversely correlated with many genes suppressed during the mature growth phase. Among them, plant-specific NAM/ATAF/CUC (NAC) transcription factors represent an interesting gene family due to their key role in the biological processes in plant development and stress responses (Jensen et al., 2014). Five NAC genes were selected for functional characterization as key factor candidates of the major transcriptome reprogramming during grapevine development. VvNAC11, VvNAC13, VvNAC33 and VvNAC60 were identified as ‘switch’ genes in the above-mentioned analysis whereas VvNAC03 was selected because it is a close homologue of tomato NOR (non-ripening), known for its crucial role in tomato fruit ripening regulation (Giovannoni, 2004; Giovannoni et al., 1995). Firstly, the five transcription factors were transiently over-expressed in Vitis vinifera to get an overview of their primary effects on native species. Secondly, we obtained grapevine plants that were stably transformed with VvNAC33 and VvNAC60 and subjected to molecular/phenotypic characterizations. VvNAC33 seemed to be involved in negative regulation of photosynthesis since over-expressing leaves revealed a chlorophyll breakdown, while VvNAC60 affected regular plant development, showing a slight growth and earlier stem lignification in comparison to a same-age plant control. These results reflected typical behaviors of plants undergoing ripening and senescence, thus supporting our working hypothesis proposing a crucial role of NACs in the transition from vegetative to mature development in grapevine. In order to identify downstream targets of the NAC transcription factors analyzed in this work, we performed microarray analysis on leaves of transient and stable ectopic expressing plants. We noted that both over-expressions affected a wide range of cellular processes and among the most represented functional categories we found transport, secondary metabolism and transcription factor activity. The identification of VvMYBA1, a known grapevine regulator of the anthocyanin biosynthetic pathway (Kobayashi et al., 2002), as VvNAC60 target suggests a VvNAC60 role in processes like anthocyanin biosynthesis featured by grape berries at the onset of ripening. Another approach used to clarify NACs roles was to check the ability of VvNACs to fulfil the tomato NOR function. Preliminary results revealed that VvNAC03 and VvNAC60 could partially complement the nor mutation in tomato, establishing a partial ripening phenotype in fruits. Taken together, these findings suggest the ability of the selected VvNACs to affect the expression of genes involved in the regulatory network that controls the developmental shift to a mature phase in grapevine. This work has shed some light on the roles of these NACs in grapevine development, but further analysis must be conducted to fully elucidate the molecular machinery in this complex regulation system.

The original objectives are listed below: 1. Design vectors for constitutive expression of endochitinase from Trichoderma harzianum strain P1. Design vectors with signal peptides to target gene expression. 2. Extend transformation/regeneration technology to other cultivars of importance in the U.S. and Israel. 3. Transform cultivars with the endochitinase constructs developed as part of objective 1. A. Characterize foliar powdery mildew resistance in transgenic plants. Background of the topic Conventional breeding of grapevines is a slow and imprecise process. The long generation cycle, large space requirements and poor understanding of grapevine genetics prevent rapid progress. There remains great need to improve existing important cultivars without the loss of identity that follows from hybridization. Powdery mildew (Uncinula necator) is the most important fungal pathogen of grapevines, causing economic losses around the world. Genetic control of powdery mildew would reduce the requirement for chemical or cultural control of the disease. Yet, since the trait is under polygenic control, it is difficult to manipulate through hybridization and breeding. Also, because grapevines are heterozygous and vegetatively propagated cultivar identity is lost in the breeding process. Therefore, there is great need for techniques to produce transgenic versions of established cultivars with heterologous genes conferring disease resistance. Such a gene is now available for control of powdery mildew of grapevines. The protein coded by the Endochitinase gene, derived from Trichoderma harzianum, is very effective in suppressing U. necator growth. The goal of this proposal is to develop transgenic grapevines with this antifungal gene, and to test the effect of this gene on resistance to powdery mildew. Conclusions, achievements and implications Gene transfer technology for grape was developed using commercial cultivars for both wine and table grapes. It paved the way for a new tool in grapevine genetic studies enabling the alteration of specific important traits while maintaining the essential features of existing elite cultivars. Regeneration and transformation technologies were developed and are currently at an advanced stage for USA wine and Israeli seedless cultivars, representing the cutting edge of grape genetic engineering studies worldwide. Transgenic plants produced are tested for powdery mildew resistance in greenhouse and field experiments at both locations. It is our ultimate goal to develop transgenic grapes which will be more efficient and economical for growers to produce, while also providing consumers with familiar products grown with reduced chemical inputs.

Rugose wood is a complex disease of grapevines, which occurs in all growing areas. The disease is spread in the field by vector transmission (mealybugs). At least five elongated-phloem- limited viruses are implicated in the various rugose wood disorders. The most fully characterized of these are Grapevine virus A (GV A) and GVB, members of a newly established genus, the vitivirus. GVC, a putative vitivirus, is much less well characterized than GV A or GVB. The information regarding the role of GVC in the etiology and epidemiology of rugose wood is fragmentary and no sequence data for GVC are available. The proposed research is aimed to study the etiology and epidemiology of rugose wood disease, and to construct genetically engineered virus-resistant grapevines. The objectives of our proposed research were to construct transgenic plants with coat protein gene sequences designed to induce post-transcriptional gene silencing (pTGS); to study the epidemiology and etiology of rugose wood disease by cloning and sequencing of GVC; and surveying of rugose wood- associated viruses in Californian and Israeli vineyards. In an attempt to experimentally define the role of the various genes of GV A, we utilized the infectious clone, inserted mutations in every ORF, and studied the effect on viral replication, gene expression, symptoms and viral movement. We explored the production of viral RNAs in a GV A-infected Nicotiana benthamiana herbaceous host, and characterized one nested set of three 5'-terminal sgRNAs of 5.1, 5.5 and 6.0 kb, and another, of three 3'-terminal sgRNAs of 2.2, 1.8 and 1.0 kb that could serve for expression of ORFs 2-3, respectively. Several GV A constructs have been assembled into pCAMBIA 230 I, a binary vector which is used for Angrobacterium mediated transformation: GV A CP gene; two copies of the GV A CP gene arranged in the same antisense orientation; two copies of the GV A CP gene in which the downstream copy is in an antigens orientation; GV A replicase gene; GV A replicase gene plus the 3' UTR sequence; and the full genome of GV A. Experiments for transformation of N. benthamiana and grapevine cell suspension with these constructs have been initiated. Transgenic N. benthamiana plants that contained the CP gene, the replicase gene and the entire genome of GV A were obtained. For grapevine transformation, we have developed efficient protocols for transformation and successfully grapevine plantlets that contained the CP gene and the replicase genes of GV A were obtained. These plants are still under examination for expression of the trans genes. The construction of transgenic plants with GV A sequences will provide, in the long run, a means to control one of the most prevalent viruses associated with grapevines. Our many attempts to produce a cDNA library from the genome of GVC failed. For surveying of rugose wood associated viruses in California vineyards, samples were collected from different grape growing areas and tested by RT-PCR for GV A, GVB and GVD. The results indicated that some of the samples were infected with multiple viruses, but overall, we found higher incidence of GVB and GV A infection in California vineyards and new introduction varieties, respectively. In this research we also conducted studies to increase our understanding of virus - induced rootstock decline and its importance in vineyard productivity. Our results provided supporting evidence that the rootstock response to virus infection depends on the rootstock genotype and the virus type. In general, rootstocks are differ widely in virus susceptibility. Our data indicated that a virus type or its combination with other viruses was responsible in virus-induced rootstock decline. As the results showed, the growth of the rootstocks were severely affected when the combination of more than one virus was present.

Grapevine is perhaps the most widely grown fruit crop. To understand the genetic make-up so as to improve the yield and quality of grapes and grape products, researchers in Europe have recently sequenced the genomes of Pinot noir and its inbred. As expected, function of many grape genes is unknown. Functional genomics studies have become the major focus of grape researchers and breeders. Current genetic approaches for gene function studies include mutagenesis, crossing and genetic transformation. However, these approaches are difficult to apply to grapes and takes long periods of time to accomplish. It is thus imperative to seek new ways for grape functional genomics studies. Virus-induced gene silencing (VIGS) offers an attractive alternative for this purpose and has proven highly effective in several herbaceous plant species including tomato, tobacco and barley. VIGS offers several advantages over existing functional genomics approaches. First, it does not require transformation to silence a plant gene target. Instead, it induces silencing of a plant gene through infection with a virus that contains the target gene sequence, which can be accomplished within a few weeks. Second, different plant genes can be readily inserted into the viral genome via molecular cloning and functions of a large number of genes can be identified within a short period of time. Our long-term goal of this research is to develop VIGS-based tools for grapevine functional genomics, made of the genomes of Grapevine virus A (GVA) from Israel and Grapevine rupestris stem pitting-associated virus (GRSPaV) from Canada. GVA and GRSPaV are members of the Flexiviridae. Both viruses have single-stranded, positive sense RNA genomes, which makes them easy to manipulate genetically and excellent candidates as VIGS vectors. In our three years research, several major breakthroughs have been made by the research groups involved in this project. We have engineered a cDNA clone of GVA into a binary vector that is infectious upon delivery into plantlets of micropropagated Vitis viniferacv. Prime. We further developed the GVA into an expression vector that successfully capable to silence endogenous genes. We also were able to assemble an infectious full-length cDNA clones of GRSPaV. In the following sections Achievements and Detailed description of the research activities, we are presenting the outcome and results of this research in details.

Original objectives . 1. Test anti-fungal gene products for activity against Uncinula necator, Aspergillus niger, Rhizopus stolonifer and Botrytis cinerea. 2. For Agrobacterium transformation, design appropriate vectors with gene combinations. 3. Use biolistic bombardment and Agrobacterium for transformation of important cultivars. 4. Characterize gene expression in transformants, as well as level of powdery mildew and Botrytis resistance in foliage of transformed plants. Background The production of new grape cultivars by conventional breeding is a complex and time-consuming process. Transferring individual traits via single genes into elite cultivars was proposed as a viable strategy, especially for vegetatively propagated crops such as grapevines. The availability of effective genetic transformation procedures, the existence of genes able to reduce pathogen stress, and improved in vitro culture methods for grapes, were combined to serve the objective of this proposal. Effective deployment of resistance genes would reduce production costs and increase crop quality, and several such genes and combinations were used in this project. Progress The efficacy of two-way combinations of Trichoderma endochitinase (CHIT42), synthetic peptide ESF12 and resveratrol upon the control of growth of Botrytis cinerea and Penicillium digitatum were evaluated in vitro. All pairwise interactions were additive but not synergistic. Per objective 2, suitable vectors with important gene combinations for Agrobacterium transformation were designed. In addition, multiple gene co-transformation by particle bombardment was also tested successfully. In New York, transformation work focused on cultivars Chardonnay and Merlot, while the technology in Israel was extended to 41B, R. 110, Prime, Italia, Gamay, Chardonnay and Velika. Transgenic plant production is summarized in the appendix. Among plants developed in Israel, endochitinase expression was assayed via the MuchT assay using material just 1-5 days after co-cultivation. Plants of cv. Sugraone carrying the gene coding for ESF12, a short anti-fungal lytic peptide under the control of the double 358 promoter, were produced. Leaf extracts of two plants showed inhibition zones that developed within 48 h indicating the inhibitory effect of the leaf extracts on the six species of bacteria. X fastidiosa, the causal organism of Pierce's disease, was very sensitive to leaf extracts from ESF12 transformed plants. Further work is needed to verify the agricultural utility of ESF12 transformants. In New York, some transformants were resistant to powdery mildew and Botrytis fruit rot. Major conclusions, solutions, achievements and implications The following scientific achievements resulted from this cooperative BARD project: 1. Development and improvement of embryogenesis and tissue culture manipulation in grape, while extending these procedures to several agriculturally important cultivars both in Israel and USA. 2. Development and improvement of novel transformation procedures while developing transformation techniques for grape and other recalcitrant species. 3. Production of transgenic grapevines, characterization of transformed vines while studying the expression patterns of a marker gene under the control of different promoter as the 35S CaMV in different part of the plants including flowers and fruits. 4. Expression of anti-fungal genes in grape: establishment of transgenic plants and evaluation of gene expression. Development of techniques to insert multiple genes. 5. Isolation of novel grape specific promoter to control the expression of future antimicrobial genes. It is of great importance to report that significant progress was made in not only the development of transgenic grapevines, but also in the evaluation of their potential for increased resistance to disease as compared with the non engineered cultivar. In several cases, increased disease resistance was observed. More research and development is still needed before a product can be commercialized, yet our project lays a framework for further investigations.