Regeneration of transgenic Cryptomeria japonica D. Don after Agrobacterium tumefaciens-mediated transformation of embryogenic tissue

Abstract

A genetic transformation procedure for Cryptomeria japonica was developed after co-cultivation of embryogenic tissues with the disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the visual reporter gene sgfp and two selectable marker genes, hpt and nptII. We were able to generate eight and three independent transgenic lines per gram of embryogenic tissue after selection on hygromycin and kanamycin medium, respectively. Transgenic plants were regenerated through somatic embryogenesis in 4 lines out of these 11 lines. Green fluorescent protein fluorescence was observed under fluorescent microscopy. Integration of the genes into the genome was confirmed by polymerase chain reaction analysis of embryogenic tissues and Southern blot analysis of regenerated plantlets.

Spatiotemporal gene expression profiles associated with male strobilus development in Cryptomeria japonica by suppression subtractive hybridization

“…Genetic modification might be a powerful tool to shorten the time needed to breed trees compared with traditional breeding methods because it is able to induce the favorable traits by introduction of specific genes in trees without unnecessary genetic transitions. A method for Agrobacterium tumefaciens-mediated transformation of embryogenic tissue was recently developed for C. japonica (Taniguchi et al 2008). Introduction of a male sterile trait by genetic modification of C. japonica is a viable approach to address the medical and public health problems of C. japonica polleninduced pollinosis.…”

High-efficiency Agrobacterium-mediated transformation of Cryptomeria japonica D. Don by co-cultivation on filter paper wicks followed by meropenem treatment to eliminate Agrobacterium

“…The stable transformation of conifers has been reported in several genera, such as Picea, Larix, and Pinus (Malabadi and Nataraja 2007). However, there are only 2 reports on the genetic transformation of C. japonica, which used particle bombardment and an Agrobacteriummediated method (Maruyama et al 2000;Taniguchi et al 2008), whereas the transformation efficiency levels were inadequate. Thus, we attempted to develop a more efficient C. japonica transformation method based on the Agrobacterium-mediated method reported by Taniguchi et al (2008).…”

“…However, there are only 2 reports on the genetic transformation of C. japonica, which used particle bombardment and an Agrobacteriummediated method (Maruyama et al 2000;Taniguchi et al 2008), whereas the transformation efficiency levels were inadequate. Thus, we attempted to develop a more efficient C. japonica transformation method based on the Agrobacterium-mediated method reported by Taniguchi et al (2008). Recently, it was reported that meropenem, a novel β-lactam antibiotic, is highly effective against Agrobacterium, and it improved transformation efficiencies in tobacco, tomato, rice, and apple compared with those observed by cefotaxime and carbenicillin (Li et al 2011;Ogawa and Mii 2007).…”

“…The embryogenic tissue used in the present study was an established cell line (Cj5-2-1), which was obtained and maintained according to the method described by Taniguchi et al (2008). To investigate the effects of antibiotics on the elimination of Agrobacterium and the growth of embryogenic tissues, approximately 15 mg of embryogenic tissue was placed at 10 positions in the maintenance medium (MM; Taniguchi et al 2008), which was supplemented with meropenem (meropenem trihydrate; Wako, Japan) at 5, 10, 20, or 40 mg l −1 or with carbenicillin sodium salt (Wako, Japan) at 250 mg l −1 , of the conventional method (Taniguchi et al 2008).…”

“…To investigate the effects of antibiotics on the elimination of Agrobacterium and the growth of embryogenic tissues, approximately 15 mg of embryogenic tissue was placed at 10 positions in the maintenance medium (MM; Taniguchi et al 2008), which was supplemented with meropenem (meropenem trihydrate; Wako, Japan) at 5, 10, 20, or 40 mg l −1 or with carbenicillin sodium salt (Wako, Japan) at 250 mg l −1 , of the conventional method (Taniguchi et al 2008). Each treatment comprised 3 replicate plates, which were incubated at 25°C for 2 weeks in the dark.…”