Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics

We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice (Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance (hpt), green fluorescent protein (gfp) and [bêta]-glucuronidase (gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per cocultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T0 plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T1 progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.

Bibliographic Details

Main Authors:	Sallaud, Christophe, Meynard, Donaldo, Van Boxtel, Jos, Gay, Céline, Bes, Martine, Brizard, Jean-Paul, Larmande, Pierre, Ortega, D., Raynal, Monique, Portefaix, Murielle, Ouwerkerk, Pieter B.F., Rueb, S., Delseny, Michel, Guiderdoni, Emmanuel
Format:	article biblioteca
Language:	eng
Subjects:	F30 - Génétique et amélioration des plantes, Oryza sativa, génie génétique, gène, adn, mutation, mutagène, méthode d'amélioration génétique, transfert de gène, http://aims.fao.org/aos/agrovoc/c_5438, http://aims.fao.org/aos/agrovoc/c_15974, http://aims.fao.org/aos/agrovoc/c_3214, http://aims.fao.org/aos/agrovoc/c_2347, http://aims.fao.org/aos/agrovoc/c_5014, http://aims.fao.org/aos/agrovoc/c_5012, http://aims.fao.org/aos/agrovoc/c_1079, http://aims.fao.org/aos/agrovoc/c_24846,
Online Access:	http://agritrop.cirad.fr/514145/ http://agritrop.cirad.fr/514145/1/514145.pdf
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