Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency

Abstract

An efficient Agrobacterium-mediated durum wheat transformation system has been developed for the production of 121 independent transgenic lines. This improved system used Agrobacterium strain AGL1 containing the superbinary pGreen/pSoup vector system and durum wheat cv Stewart as the recipient plant. Acetosyringone at 400 μM was added to both the inoculation and cultivation medium, and picloram at 10 mg l−1 and 2 mg l−1 was used in the cultivation and induction medium, respectively. Compared with 200 μM in the inoculation and cultivation media, the increased acetosyringone concentration led to significantly higher GUS (β-glucuronidase) transient expression and T-DNA delivery efficiency. However, no evident effects of acetosyringone concentration on regeneration frequency were observed. The higher acetosyringone concentration led to an improvement in average final transformation efficiency from 4.7% to 6.3%. Furthermore, the concentration of picloram in the co-cultivation medium had significant effects on callus induction and regeneration. Compared with 2 mg l−1 picloram in the co-cultivation medium, increasing the concentration to 10 mg l−1 picloram resulted in improved final transformation frequency from 2.8% to 6.3%, with the highest frequency of 12.3% reached in one particular experiment, although statistical analysis showed that this difference in final transformation efficiency had a low level of significance. Stable integration of foreign genes, their expression, and inheritance were confirmed by Southern blot analyses, GUS assay, and genetic analysis. Analysis of T1 progeny showed that, of the 31 transgenic lines randomly selected, nearly one-third had a segregation ratio of 3:1, while the remainder had ratios typical of two or three independently segregating loci.

High Efficiency Wheat Transformation Mediated by Agrobacterium tumefaciens

“…Embryos and cultured cells of several wheat genotypes were tested, and various factors were examined in the early 2000s, however, the frequency of transformation was mostly less than 5 % of the inoculated tissue pieces. Even in the recent reports (He et al 2010 ;Bińka et al 2012 ), the frequency of transformation in wheat did not change much from those described in the early reports.…”

Agrobacterium mediated transformation of indica rice (Oryza sativa L.) for insect resistance

“…In the present study, a large number of rice plants carrying Cry1C gene has been produced in IR64 cultivar by Agrobacterium mediated transformation method. High transformation efficiency was achieved in this study due to the use of super virulent LBA4404 (pCAMBIA1305.1/ Cry1C) in IR64 cultivar (He et al 2010) and by incubating the co-cultivated calli in hygromycin free medium for 1 week before transferring to selection medium.…”

Wheat Biotechnology: Current Status and Future Prospects

“…Biolistics can also be utilised to deliver DNA into the genomes of mitochondria and chloroplasts. Perceived advantages, particularly in terms of lower copy number have driven significant research into using Agrobacterium tumefaciens (synonym Rhizobium radiobacter) for the DNA transfer process, and many laboratories now report successful wheat transformation using Agrobacterium (Binka et al 2012;Campa et al 2005;Cheng et al1997;Cheng et al 2003;Ding et al 2009;Guo et al 1998;Haliloglu and Baenziger 2003;Hamid et al 2012;He et al 2010;Jones et al 2005;Khanna and Daggard 2003;Mahalakshmi and Khurana 1995;Marks et al 1989;McCormac et al 1998;Mitic et al 2004;Mooney et al 1991;Murin et al 2011;Parrott et al 2002;Patnaik et al 2006;Pérez-Piñeiro et al 2012;Przetakiewicz et al 2004;Pukhalskii et al 1996;Rashid et al 2012;Shi et al 2011;Song et al 2012;TamasNyitrai et al 2012;Trifonova et al 2001;Wu et al 2003Wu et al , 2008Xia et al 1999). One particular unpublished method developed by the Japan Tobacco company (PureWheat Technology) has been licensed to specific laboratories who report wheat transformation efficiencies of more than 50 %.…”