Transgenic Pinus radiata from Agrobacterium tumefaciens?mediated transformation of cotyledons

Abstract

A method for Agrobacterium tumefaciens-mediated transformation of Pinus radiata cotyledon explants was developed using commercially available open-pollinated seed. Pinus radiata is the most widely planted commercial conifer species in the Southern Hemisphere. Reports on transformation of this species have relied on particle bombardment of embryogenic callus derived from immature embryos. The main drawback to the method is the small number of genotypes that are amenable to transformation and regeneration. Since more than 80% of genotypes of radiata pine can be regenerated using cotyledons from mature seed, cotyledon explants were cocultivated with A. tumefaciens strain AGL1 containing a plasmid coding for the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (GUS) gene (uidA). Transformed shoots were selected using either geneticin or kanamycin. Critical factors for successful transformation were survival of the cotyledons after cocultivation and selection parameters. Of the 105 putative transformants that were recovered from selection media, 70% were positive for integration of the nptII gene when analysed by PCR. GUS histochemical assay for uidA expression was unreliable because of reaction inhibition by unidentified compounds in the pine needles. Further, only 4 of the 26 independent transformants characterised by PCR and Southern analysis contained an intact copy of both genes. The remaining 22 transformants appeared to have a truncated or rearranged copy of the T-DNA. It is possible that the truncation/rearrangements are due to the Cauliflower mosaic virus (CaMV) 35S promoter. Analysis of the T-DNA junction sites and sequencing of the introduced DNA will help elucidate the nature of T-DNA insertion so that genetic modification of radiata pine can be targeted effectively.

Agrobacterium-mediated Transformation via Somatic Embryogenesis System in Korean fir (Abies koreana Wil.), A Korean Native Conifer

“…These RNA samples were converted into cDNA, and quantitative real-time RT-PCR was performed followed by Grant et al (2004 …”

“…radiate (Grant et al, 2004). Although currently several methods exist for transferring foreign genes into plants, but somatic embryogenesis has proved to be the best method to produce transgenic plants in a number of conifer species that ultimately leads to their application in commercial forestry.…”

“…4). The bacterial nptII gene is the most frequently used selectable marker gene for generating transgenic plants in conifers (Klimaszewska et al, 1997(Klimaszewska et al, , 2001Grant et al, 2004;. The nptII gene confers kanamycin resistance by inhibiting protein synthesis and is a conditional positive selection system that is toxic to untransformed cells (Miki and McHugh, 2004).…”

“…However, conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency (Grant et al, 2004;Find et al, 2005). Moreover, most of gymnosperm transformation protocols was established in non-Korean native conifers Klimaszewska et al, 2001;Nigro et al, 2004) which are difficult to apply in Korean species.…”

Genome engineering of woody plants: past, present and future

“…Although softwoods are more recalcitrant to Agrobacterium-mediated transformation, improvements in tissue culture systems have allowed the production of transgenic softwoods, Pinus radiata [26], Pinus taeda [27,28], and Picea abies [27]. As in the case of Populus and Eucalyptus plants, the introduction and overexpression of genes of commercial interest have also been demonstrated, e.g., production of salt-tolerant Pinus taeda [29].…”