Transformation of American Chestnut (Castanea dentata (Marsh.) Borkh) Using RITA® Temporary Immersion Bioreactors and We Vitro Containers

Abstract

American chestnut (Castanea dentata (Marsh.) Borkh) was almost completely wiped out by the fungal pathogen, Cryphonectria parasitica (Murrill) M.E. Barr. Another invasive pathogen, Phytophthora cinnamomi Rands, is devastating American chestnuts in the southern region of the United States. An alternative approach for controlling these pathogens is to use genetic engineering or gene editing. We successfully transformed American chestnut with a detoxifying enzyme, oxalate oxidase, to enhance blight tolerance and more recently with the Cast_Gnk2-like gene, which encodes for an antifungal protein, to be tested for P. cinnamomi putative tolerance. Eight somatic embryo lines were transformed using three methods of selection: semisolid medium in Petri plates, liquid medium in RITA® temporary immersion bioreactors, or liquid medium in We Vitro containers. No significant differences were found between the treatments. These methods will allow for further testing of transgenes and the development of enhanced pathogen resistance in chestnut. It can serve as a model for other tree species threatened by invasive pests and pathogens.

Pathogen‐induced expression of a blight tolerance transgene in American chestnut

“…The win3.12‐OxO vector (Figure 1) was constructed with the win3.12 promoter from poplar and the oxalate oxidase ( OxO ) gene from wheat and transformed into American chestnut somatic embryos (McGuigan et al, 2020). Twenty‐two independent transgenic embryo lines were recovered and tested for insert copy number using quantitative PCR (qPCR).…”

“…Somatic embryos from the wild‐type C. dentata ‘Ellis 1’ (Ellis) clonal line were used for Agrobacterium‐ mediated transformation (McGuigan et al, 2020; Polin et al, 2006; Zhang et al, 2011). Somatic embryos were co‐cultivated with Agrobacterium containing the pwin3.12‐OxO vector and all transformed embryos were selected using paromomycin.…”

“…Transgenic events were designated with the prefix WX (W = win3.12 X = OxO ). Shoots were regenerated from transformed somatic embryos (McGuigan et al, 2020) and maintained in multiplication medium until elongated for rooting (Oakes et al, 2016). Potted plantlets were generated from tissue‐cultured shoots using ex vitro rooting techniques and acclimatized in high‐humidity growth chambers (Oakes et al, 2016).…”

Genetic Transformation of Quercus ilex Somatic Embryos with a Gnk2-like Protein That Reveals a Putative Anti-Oomycete Action

“…Cucumber transgenic plants overproducing the Gnk2-1 gene have displayed resistance to Fusarium oxysporum [ 34 ]. McGuigan et al [ 35 ] were recently successful in overexpressing the Cast_Gnk2-like gene in American chestnut somatic embryos, but the tolerance of transgenic plants to P. cinnamomi has not yet been checked.…”

Application of Tissue Culture in Plant Reproduction

“…The researchers concluded that the root rot resistance locus PaLAR3B is successfully delivered from elite Norway spruce parent trees to their SE progeny through the SE-propagation method and there is no trade-off between root rot resistance locus PaLAR3B and somatic embryo production ability. McGuigan et al [7] used SE in combination with genetic transformation to confer resistance to Cryphonectria parasitica and P. cinnamomi in American chestnut. Somatic embryos were successfully transformed with a detoxifying enzyme, oxalate oxidase, to enhance blight tolerance, or with the Cast_Gnk2-like gene, which encodes for an antifungal protein, to be tested for putative tolerance to P. cinnamomi.…”