The Grapevine FanLeaf Virus-Coat Protein (GFLV CP) gene was inserted through Agrobacterium-mediated transformation in Vitis vinifera "Nebbiolo", "Lumassina" and "Blaufränkisch". Two plasmids were used: pGA-CP+ (full-length GFLV CP gene with an introduced start codon) and pGA-AS (same gene in antisense orientation). Forty-three transgenic lines were regenerated. As several lines in Southern blots share same hybridization patterns, eight independent line groups resulted for "Nebbiolo", one for "Lumassina", and two for "Blaufränkisch". Inserted T-DNA copies ranged from one to three; one line probably contains an incomplete copy of T-DNA. Except for one "Nebbiolo" line, no evidence for methylation of the transgene at cytosine residues was found by Southern analyses. Specific mRNA was present at variable expression levels; some lines accumulated the coat protein while in others the protein was not detectable by ELISA.

In clonal unisexual vertebrates, the genes specifying the males become dispensable. To study the fate of such genes the gynogenetic all-female fish Poeciiaformosa was treated with androgens. Phenotypic males were obtained that exhibited the complete set of male characteristics of closely related gonochoristic species, including body proportions, pigmentation, the extremely complex insemination apparatus of poeciliid fish, sexual behavior, and spermatogenesis. The apparent stability of such genic structures, including those involved in androgen regulation, is contrasted by high instability of noncoding sequences. Frequent mutations, their donal transmission, and at least two truly hypervariable loci leading to individual differences between these otherwise donal organisms were detected by DNA fingerprinting. These observations substantiate the concept that also in "ameiotic" vertebrates certain compartments of the genome are more prone to mutational alterations than others.Clonal organisms occur in natural populations of multicellular animals at all levels of organismic evolution, but within vertebrates they are confined to teleost fish, amphibians, and reptiles (1). They exist as unisexual lineages that exclude effective genetic recombination. In such animals the genetic information specifying the other sex has apparently become dispensable. These organisms are therefore uniquely suited to investigate questions centering on problems of "dispensable" genes (2). Moreover, the existence of naturally occurring clonal animals poses questions about their origin and abilities to evolve in the absence of meiotic recombination.The amazon molly, Poecilia formosa, is an all-female poeciliid fish that inhabits freshwater streams and brackish coastal lagoons over a broad geographic range from southeastern Texas to northeastern Mexico. The reproductive mechanism in P. formosa is gynogenesis, a modified form of parthenogenesis. Sperm do not participate in syngamy but are required to trigger the onset of embryogenesis in diploid eggs, containing only the maternal genetic information (3, 4). Except for rare cases (5) the offspring are clonal replicates of their mothers (6-8). In natural populations sperm is contributed by males of two closely related bisexual species that occur sympatric with P. formosa. In the northern range this is Poecilia latipinna (5), whereas in the mexican habitats it is Poecilia mexicana (10). Biochemical and cytological data (8) supported the initial hypothesis (11) that the amazon molly is a hybrid species between P. latipinna and P. mexicana. The question arose if the genes determining male sex, phenotype, and behavior are absent or not functional in P. formosa.Preliminary studies (12-15) reported that treatment with androgenic steroids induces several phenotypic changes that resembled the male characteristics of related species. Androgen-induced masculinization, therefore, appeared to be a useful approach to investigate if the genes instrumental in determining the male phenotype and behavio...

A collection of 127 putatively transgenic individuals of Vitis vinifera cv. Russalka was characterized by PCR and Southern hybridization. Six different constructs containing the neomycin phosphotransferase (nptII) marker gene and sequences of the Grapevine Fanleaf Virus Coat Protein (GFLV CP) gene including non-translatable and truncated forms were transferred via Agrobacterium-mediated transformation. Detection of transgenic sequences by PCR was positive in all lines. Southern blot analysis revealed that the number of inserted T-DNA copies ranged from 1 to 6. More than 46% of the tested transgenic lines contain one copy of the inserted T-DNA, qualifying them as interesting candidates for further breeding programs. Southern data of one line indicate the presence of an incomplete copy of the T-DNA, thus confirming previous PCR results. Since many putative transgenic lines shared identical hybridization patterns, they were clustered into 39 lines and considered as having originated from independent transformation events. The detection of the tetracycline (TET) resistance genes in 15% of the lines shows that an integration of plasmid backbone sequences beyond the T-DNA borders occurred. Enzyme-linked immunosorbent assay (ELISA) performed on leaf tissue did not show any accumulation of the GFLV CP in the 39 transgenic lines analyzed. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot were carried out; RT-PCR analyses showed that the GFLV CP mRNA was expressed at variable levels.

Mutations of the tyrosinase gene are one common cause of a similar phenotype in all vertebrates, known as albinism. In an attempt to contribute to an understanding of the genetic hierarchy governing the development of pigmentation, we have used a mouse tyrosinase minigene under the control of its 5.2 kb upstream promoter region to rescue two different albino mutations in the medakafish, Oryzias latipes. Around hatching stages an almost perfect phenocopy of the wildtype pigmentation was obtained. Subsequent ectopic melanin overproduction indicated a possible incompatibility of the heterologous mouse promoter for stable expression during the entire ontogenesis. Like in some tyrosinase transgenic mouse lines a strong variegation effect was observed. The transgene-mediated pigmentation phenotype was obtained up to the eighth offspring generation. The phenotypic effects of the tyrosinase transgene in different albino mutant strains places the i3-locus upstream and the b-locus downstream of the tyrosinase locus i1 in the genetic hierarchy leading to wildtype pigmentation.

Attempts to conserve and utilise autochthonous grapevine germplasm in modern breeding programmes, are sometimes faced with the challenge that virus-free plants of old grapevine varieties and clones are hard to find. From 50 year-old vineyards in Frankonia the Vitis vinifera cv. Domina was selected showing particularly interesting loose-bunch architecture with fewer berries. However this valuable germplasm was carrying an Arabis mosaic virus (ArMV) infection requiring a reliable and effective method to produce healthy mother plants for clonal selection. Somatic embryogenesis was established from anthers as the most promising technical approach. The absence of ArMV in 46 regenerated plant lines was confirmed by ELISA and IC-RT PCR, repeated after different time intervals in vitro and in vivo after acclimatisation, and after one dormancy period under glasshouse conditions. Morphologically, all grapevines appeared true-to-type, and a screening of 20 plants by flow cytometry to determine the ploidy level and to exclude the risk of undesired genetic variability confirmed that all tested plants were diploid. Field evaluations of the initially selected bunch traits are currently underway.Keywords Biodiversity . Fanleaf disease . Vitis vinifera . Tissue culture . ELISA . IC-RT-PCR . Ploidy level etiology of fanleaf disease of grapevines in central