Exploring Organogenesis in American Chestnut

Abstract

American chestnut (Castanea dentata) was once a major presence in the forests of the Eastern United States. However, fifty years after chestnut blight, a canker disease caused by Cryphonectria parasitica, was first observed in the Bronx Zoo in 1904, the tree was considered functionally extinct. Efforts to increase disease resistance in the species via genetic engineering are ongoing, however they would be aided by improvements in the efficiency and speed of in vitro regeneration. To begin optimizing a de novo organogenesis regeneration protocol for C. dentata, we tested seven different callus induction media formulations as well as Agrobacterium-mediated transformation of explants with ten different morphogenic regulators (MRs) that had been reported to increase shoot induction when overexpressed in other plant species. We identified a high-performing callus induction media (WPM with 2 mg BAP and 0.5 mg NAA) that produced callus on 87.5% of C. dentata leaf explants after being kept in a dark environment for 3 weeks. In our three transformation experiments with MRs, while we found some MRs to cause significantly more callus to form than others, we found no significant improvement in callus induction in explants transformed with MRs compared to GFP-transformed controls. In some cases, the GFP-transformed control performed significantly better. Across both studies, four adventitious shoots were observed. These represent the first reported shoots formed by C. dentata through de novo organogenesis and give us optimism that with more optimization, a consistent protocol can be generated.