Hairy root production in Arabidopsis thaliana: cotransformation with a promoter-trap vector results in complex T-DNA integration patterns

Citation
M. Karimi et al., Hairy root production in Arabidopsis thaliana: cotransformation with a promoter-trap vector results in complex T-DNA integration patterns, PL CELL REP, 19(2), 1999, pp. 133-142
Citations number
37
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Plant Sciences","Animal & Plant Sciences
Journal title
PLANT CELL REPORTS
ISSN journal
0721-7714 → ACNP
Volume
19
Issue
2
Year of publication
1999
Pages
133 - 142
Database
ISI
SICI code
0721-7714(199912)19:2<133:HRPIAT>2.0.ZU;2-2
Abstract
In comparison with the production of transgenic plants, the generation of h airy roots has the advantage that more independent transgenic lines can be produced in a shorter period of time. Therefore, we wanted to combine this approach with the promoter-trapping strategy to identify nematode-induced p lant promoters. For the efficient production and culture of transgenic hair y root lines of Arabidopsis thaliana, the standard Agrobacterium rhizogenes transformation procedure was modified to avoid rapid callusing of the hair y roots. An average of 0.72 independent kanamycin-resistant (Km(R)) roots w ere obtained per leaf piece. However, a much lower frequency of reporter ge ne activation was obtained than expected from experiments with the same vec tors in Agrobacterium tumefaciens: of more than 700 independent Km(R) hairy roots tested, only 8 were beta-glucuronidase (GUS) positive. DNA hybridiza tion was done on ten hairy root lines, of which one had a single truncated T-DNA and the others multiple copies of T-DNA that led to complex hybridiza tion patterns. In a parallel analysis of A. thaliana plants transformed wit h the same vectors using A. tumefaciens, relatively simple T-DNA integratio n patterns were obtained. The low occurrence of GUS-positive hairy root lin es in our experiments could be explained by the multiple T-DNA copies, espe cially in inverted array, that result in high frequencies of gene inactivat ion.