Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs, plant ecotypes and Agrobacterium strains

Abstract

The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the ß-glucuronidase (GUS) reporter gene showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80-90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.

PLETHORA Genes Control Regeneration by a Two-Step Mechanism

“…2 A-D). Shoots were regenerated from all of the tested in wild type explants as previously reported [4,18]. Shoot regeneration was completely abolished in plt3; plt5-2; plt7 tissue ( Fig.…”

A Combinatorial Interplay Among the 1-Aminocyclopropane-1-Carboxylate Isoforms Regulates Ethylene Biosynthesis inArabidopsis thaliana

“…The hygromycine resistance gene was isolated by PCR using pIG121-Hm as template (AKAMA et al 1992) and was subcloned as a NcoI (blunted) / EcoRV fragment into the BglII (blunted) sites of pPZP122.…”

Establishment of bispyribac selection protocols for <i>Agrobacterium tumefaciens</i>- and <i>Agrobacterium rhizogenes</i>-mediated transformation of the oil seed plant <i>Jatropha curcas</i> L.

“…e binary vectors pIG121-Hm (kanamycin and hygromycin selection; Ohta et al 1990, Akama et al 1992, pBIN30 (bar selection; Miyashima et al 2011), and A2 (bispyribac selection, PalSelect system, Kumiai Chemical Industry, Tokyo, Japan) were introduced into A. tumefaciens strain LBA4404 and used for stable transformation. To construct the A2-IG vector, an intron-containing β-glucuronidase (gus) gene expression cassette was ampli ed from pIG121-Hm by PCR using the primers 5′-AAG CTT GCA TGC CTG CAGG-3′ (forward) and 5′-CCG ATC TAG TAA CAT AGA TGAC-3′ (reverse).…”