Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation

Abstract

The availability of a system for direct transfer of anti-fungal candidate genes into American chestnut (Castanea dentata), devastated by a fungal blight in the last century, would offer an alternative or supplemental approach to conventional breeding for production of chestnut trees resistant to the blight fungus and other pathogens. By taking advantage of the strong ability of embryogenic American chestnut cultures to proliferate in suspension, a high-throughput Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into the tree was established. Proembryogenic masses (PEMs) were co-cultivated with A. tumefaciens strain AGL1 harboring the plasmid pCAMBIA 2301, followed by stringent selection with 50 or 100 mg/l Geneticin. A protocol employing size-fractionation to enrich for small PEMs to use as target material and selection in suspension culture was applied to rapidly produce transgenic events with an average efficiency of four independent transformation events per 50 mg of target tissue and minimal escapes. Mature somatic embryos, representing 18 transgenic events and derived from multiple American chestnut target genotypes, were germinated and over 100 transgenic somatic seedlings were produced and acclimatized to greenhouse conditions. Multiple vigorous transgenic somatic seedlings produced functional staminate flowers within 3 years following regeneration.

Genomics of Fagaceae

“…In the final generation, only highly resistant trees are left in the seed orchard. The diagram has been modified from the one designed by John Frampton at North Carolina State University, Raleigh, NC, USA (Andrade and Merkle 2005;Powell et al 2006;Merkle et al 2007;Maynard et al 2008;Andrade et al 2009). Significant support for transgenic chestnut research is now being provided by the FHI, sponsored by the USDA Forest Service, Duke Energy, and the US Endowment for Forestry and Communities (http://www.foresthealthinitiative.org).…”

High-efficiency Agrobacterium-mediated transformation in Quercus robur: selection by use of a temporary immersion system and assessment by quantitative PCR

“…Torreblanca et al (2010) maintained the selection pressure during the entire regeneration process (including embryo maturation and germination), to prevent the formation of chimaeric olive transgenic embryos, and kanamycin has been used both in proliferation and rooting medium of transgenic green ash (Du and Pijut 2009) and poplar (Yevtushenko and Misra 2010). However, transgenic embryos of other woody plants have also been matured and germinated in the absence of selective agents, as reported for avocado (Palomo-Ríos et al 2012), rubber tree (Leclercq et al 2010), and other species of Fagaceae such as European and American chestnut , Andrade et al 2009) and cork oak Ordás 2007, Álvarez et al 2009). Although no chimaeric oak transgenic embryos have been detected with the selection pressure described in this study, this problem may be more common than originally thought (Flachowsky et al 2008, Padilla andBurgos 2010), and establishing the concentrations of antibiotics that can be applied to transgenic materials in each step of their development is of a practical interest.…”

“…Cell suspension cultures have been used during the selection phase in mango (Mathews et al 1992), avocado (Cruz-Hernandez et al 1998;Raharjo et al 2008) and olive (Torreblanca et al 2010), although no clear comparison was made with selection on semi-solid medium. An efficient transformation protocol involving the use of embryogenic suspension cultures throughout the whole procedure has been established for American chestnut (Fagaceae) (Andrade et al 2009). In addition to the increased proliferation rates and the stringent selection conditions provided by liquid medium, use of the TIS provides other advantages, such as easier handling of large amounts of starting material, and removal of phenolic compounds released by the explants after transformation.…”

“…The difficulty in identifying each transformation event within all culture flasks has been identified as a limiting step in transformation experiments involving cell suspension cultures. All of the transformed material developed in a flask must be considered as a unique event because of the characteristics of the culture (Andrade et al 2009). Therefore, the TIS potentially yields more transformed lines with the same starting material, which considerably reduces the space, labour and costs involved in the process.…”

Biotechnological efforts for preserving and enhancing temperate hardwood tree biodiversity, health, and productivity

“…Additional conditions shown to affect transformation efficiency were inoculation and pre-culture time (Álvarez and Ordás 2007;Corredoira et al 2007;Li et al 2007;Shen et al 2008;Andrade et al 2009;Du and Pijut 2009;Liu and Pijut 2010), Agrobacterium OD 600 (Álvarez and Ordás 2007;Li et al 2007;Shen et al 2008), and tissue type (Britton et al 2007;Corredoira et al 2007;Renying et al 2007;Shen et al 2008). In black locust, Shen et al (2008) noted a variation in sensitivity to antibiotics with respect to tissue type, with stems being more sensitive than cotyledons.…”