A transposon tagging strategy with Ac on plant cell level in heterologous plant species

Abstract

Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. The maize transposable element Ac can have a 'late' excision time during leaf development in certain transgenic tobacco plants. This was visualized with an assay based on Ac-excision restoring GUS-expression. Leaves of the described plants contain over 103 small blue spots, each of these spots representing an independent excision event. Leaves showing this 'late' excision phenomenon may be used for transposon tagging experiments at plant cell level. Plants which display 'late' Ac-excision do not detectably express GUS during the preceding callus phase, thus allowing transformants to be preselected for a 'late' timing of excision. To examine the applicability of this phenomenon a phenotypic selection assay for excision of Ac was used. Transformed calli containing Ac within the hygromycin resistance gene were regenerated and protoplasts isolated from leaves of regenerated plants were selected on hygromycin. Up to 0.8% of these protoplasts displayed hygromycin resistance. The hygromycin resistant derivatives analyzed were shown to represent independent transposition events. Ac-insertions which can be generated in this way may be used for transposon tagging experiments at cell level.

Transactivation of Ds by Ac-transposase gene fusions in tobacco

“…The vector pTT282 was constructed by ligating a 7.2 kb fragment of pTT218 (Hating et al 1989), obtained by SacII digestion, filling-in and BamH1 digestion, to pBI121, which had been digested with EcoRI, followed by filling in and BamHI digestion. The constructs were introduced into Agrobacterium tumefaciens LBA4404 (Hoekema et al 1983) as previously described (Rommens et al 1991 a).…”

“…Petit Havana SR-1), with Agrobaeterium strains containing the binary plasmid constructs. After infection, explants were placed on feeder layers of Petunia Albino Comanche cells for 2 days and subsequently selected on medium (Rommens et al 1991 a) containing either 100 rag/1 kanamycin (pTT269-pTT273, pTT283) or 20 mg/1 chloramphenicol (pTT265-pTT268).…”

“…However, it has been shown that Ac can transpose throughout plant development (Yoder et al 1988;Hehl et al 1990). Furthermore, it was demonstrated that the timing and frequency of transposition is variable among individual maize and tobacco plants (Fedoroff 1989;Rommens et al 1991 a). This diverse behaviour of Ac may complicate gene tagging experiments.…”

Ac-induced disruption of the doubleDs structure in tomato

“…Hybridization patterns show that the fragments are homologous to repetitive and single copy plant DNA sequences, respectively. E, EcoRI been documented in many plant species (for a review, see Haring et al 1991). Excision of doubleDs, however, could not be demonstrated in the genome of AATI915-24.…”

Characterization of theAc/Ds behaviour in transgenic tomato plants using plasmid rescue

“…[18]. DNA isolation, restriction digests, Southern blotting, hybridizations and autoradiography were performed as described previously [26].…”