The pea (Pisum sativum L.) varieties Baroness (United Kingdome) and Baccara (France) were transformed via Agrobacterium tumefaciens-mediated gene transfer with pGPTV binary vectors containing the bar gene in combination with two different antifungal genes coding for polygalacturonase-inhibiting protein (PGIP) from raspberry (Rubus idaeus L.) driven by a double 35S promoter, or the stilbene synthase (Vst1) from grape (Vitis vinifera L.) driven by its own elicitor-inducible promoter. Transgenic lines were established and transgenes combined via conventional crossing. Resveratrol, produced by Vst1 transgenic plants, was detected using HPLC and the PGIP expression was determined in functional inhibition assays against fungal polygalacturonases. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated.
Epicotyl segments and nodus expiants from etiolated seedlings of Pisum sativum were transformed using Agrobacterium tumefaciens strains GV 2260 (p35S GUS INT) and GV 3850 HPT carrying either a neomycin- or hygromycinphosphotransferase-gene as selectable markers. The transgenic character of hygromycin- or kananamycin-resistant tissue was confirmed by detection of nopaline or neomycinphosphotransferase-II- and ß-glucuronidase activity in crude extracts of resistant tissues. Up to 5 % of developing shoots from shoot proliferating nodi were regenerated via organogenesis to kanamycin-resistant plantlets. Transformation frequency in vitro was found to be influenced by expiant source, A. tumefaciens strain, pea genotype and duration of cocultivation. Acetosyringone did not increase the transformation rate.
Distribution and properties of pea (Pisum sativum L.) cells, competent for Agrobacterium-mediated transformation were analysed by in situ histochemical detection of GUS ([3-glucuronidase) activity, 4 d after inoculation with engineered Agrobacterium tumefaciens. The vector system consisted of the hypervirulent disarmed strain EHA101 and the binary plasmid pIBGUS, carrying an intron-containing, 35S-promotor driven gusA (or uidA) gene and two selectable marker genes. Cells competent for transformation were mainly restricted to the dedifferentiating cells neighbouring the vascular system of cotyledon and epicotyl explants. A standardized assay was developed, allowing determination and quantification of factors influencing number and distribution of competent cells. In etiolated seedlings, competence for transformation decreased with the distance of the epicotyl explant from the shoot apex and was specifically induced by the exogenous application of auxins. Transient expression of gusA after Agrobacterium-mediated DNA transfer was dramatically reduced upon application of cell-cycle and DNA replication inhibitors aphidicolin, colchicine and nalidixic acid. GUS expression after direct DNA transfer of double-stranded plasmid DNA (via PEG into protoplasts or via particle bombardment of epicotyl segments) was independent of cell-division/DNA replication.A GUS-positive mutant of EHA101 was constructed to allow in situ analysis of attaching bacteria within the plant tissue. Attachment and invasion was inhibited by well-developed cuticula but was restored after chloroform treatment of the tissue surface. Moreover, no correlation was found between distribution of attaching bacteria and the pattern of transformation-competent cells.
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