Genetic transformation of European chestnut

Abstract

Stable incorporation of the nptII gene into Castanea sativa Mill. has been achieved by Agrobacteriummediated transformation. The transformation assays were performed by infecting wounded hypocotyls with a strain of Agrobacterium tumefaciens, LBA 4404 harbouring the plasmid p35SGUSINT. Although two schemes of selection were tested, many escapes were obtained. The best strategy to avoid this problem is the introduction of higher concentrations of kanamycin in the culture medium, immediately after coculture. PCR analysis showed of the selectable nptII gene integration in the plant genome. β-Glucuronidase histochemical assay revealed the expression of the uidA gene in shoots, regenerated from transformed explants.

Agrobacterium rhizogenes -mediated DNA transfer to Aesculu s hippocastanum L. and the regeneration of transformed plants

“…However, it is comparable to transformation efficiencies reported for a vast majority of tree species-for example, 0.9% in apple rootstock M26 (Holefors et al 1998), 6.7% in apple rootstock M.9/29 (Zhu et al 2001), 1.5-1.8% in Castanea sativa (Seabra and Pais 1998), 7% (Confalonieri et al 2000) and 11% (Delledonne et al 2001) in Populus alba. Moore et al (1979) showed that most hairy roots emerged from the pericycle tissue of the carrot vascular cylinder.…”

Agrobacterium-mediated transformation of American chestnut (Castanea dentata (Marsh.) Borkh.) somatic embryos

“…The first report of successful Agrobacterium-mediated transformation in the Castanea genus showed transgenic shoots regenerated from European chestnut cotyledons (Seabra and Pais, 1998). This was followed by a report of the transformation of European chestnut somatic embryos that were germinated and micropropagated into whole plants (Corredoira et al, 2004).…”

Agrobacterium-mediated transformation of European chestnut embryogenic cultures

“…However, significant differences between 3 days and 4 days were only achieved with the strain/plasmid combination EHA105/pUbiGUSINT. A similar cocultivation period has been reported to be useful for other woody species, including apple (De Bondt et al 1994), chestnut (Seabra and Pais 1998), elm (Gartland et al 2000); 5 days of coculture was utilized in tea (Mondal et al 2001).…”

“…This concentration is higher than those generally employed in other embryogenic systems of woody species such as pecan (McGranahan et al 1993) and Ginkgo biloba (DuprØ et al 2000). A similar kan level was also selected by Seabra and Pais (1998) for the transformation of chestnut hypocotyl segments. The addition of 300 mg/l carbenicillin and 200 mg/l cefotaxime, which were used to avoid the overgrowth of Agrobacterium, actually had a visible beneficial effect on the growth and multiplication of chestnut somatic embryos (data not shown).…”

“…However, no transgenic somatic embryos were achieved, and only transgenic calli were produced. Subsequently, a procedure based on an organogenic system was described by Seabra and Pais (1998) for chestnut hypocotyls, but only very low transformation frequencies and chimeras were reported. In this sense, somatic embryogenesis is an important tool for plant regeneration from transformed cells since it avoids the chimerism that may occur in shoots regenerated via organogenesis (Eddo et al 2000).…”