Stable expression of the GUS reporter gene in chrysanthemum depends on binary plasmid T-DNA

Abstract

Three chrysanthemum (Dendranthema grandiflora) cultivars were cocultivated with 2 Agrobacterium tumefaciens strains in combination with 4 pBIN19 derived binary plasmids, all carrying the Nosnptll selection gene and 35Sgus(intron) reporter gene. All binary plasmids transferred DNA to chrysanthemum explants but only pMOG410 gave good stable expression of GUS. This plasmid differs from the other plasmids in 2 aspects: 1) It carries a restored nptll gene and 2) the selection gene is positioned at the left border side of the reporter gene. Cocultivation with AGLO(pMOG410) yielded up to 13 GUS positive shoots per 100 explants. The presence of the gus and nptll gene in recovered shoots was confirmed by PCR and Southern blot analysis.

Genetic engineering of chrysanthemum (Chrysanthemum morifolium): Current progress and perspectives

“…However, some barriers to the establishment of a chrysanthemum transformation system have been reported: low transformation e ciency (De Jong et al 1994;Renou et al 1993;Urban et al 1994), regeneration of chimeric plants comprising both transgenic and non-transgenic tissues (Benetka and Pavingerová 1995;Pavingerová et al 1994) and transgene inactivation, also known as gene silencing (Takatsu et al 2000). Ledger et al (1991) rst tried to generate transgenic chrysanthemum (Dendranthema indicum 'Korean') using A. tumefaciens strain LBA4404, but the transformation frequency was extremely low (1.7%).…”

“…De Jong et al (1994) and Urban et al (1994) described the di erence in transformation frequencies of chrysanthemum using di erent Agrobacterium strains and indicated that the AGL0 and EHA105 strains showed higher transformation frequencies than LBA4404. In contrast, no signi cant di erence in the transformation frequency was recognized for four cultivars, 'Shuho no chikara' (Shinoyama et al 2002a(Shinoyama et al , 2002b(Shinoyama et al , 2003, 'Yamate shiro' (Shinoyama et al 2002a(Shinoyama et al , 2008(Shinoyama et al , 2012a(Shinoyama et al , 2012b, 'Yamabiko' and 'New Summer Yellow' (Takatsu et al 2000), even if di erent Agrobacterium strains were used.…”

“…In many cases, juvenile explants are used, such as newly formed expanding leaves (Ledger et al 1991). Remarkably, De Jong et al (1994 succeeded in the transformation using pedicels that were removed from the owers of chrysanthemum plants that were nonaseptically grown in greenhouses.…”

“…De Jong et al (1994) rst succeeded in increasing the Agrobacterium infectability of chrysanthemum by adding 100 µM acetosyringone to the co-cultivation medium. Fukai et al (1995) and Takatsu et al (1998Takatsu et al ( , 2000 con rmed the e ect of acetosyringone.…”

“…Florist chrysanthemums are sensitive to kanamycin, and the application of a high concentration of kanamycin in the selection medium inhibits shoot formation (De Jong et al 1994). Other antibiotics, such as hygromycin, paromomycin and geneticin, have also been successfully used for the selection of transgenic chrysanthemums (Aida et al 2004;Renou et al 1993;Sherman et al 1998b;Shinoyama et al 1998).…”