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The American Chestnut Project

Tissue Culture and Transformation

beaker with liquid in itPlant tissue culture (micropropagation) is a method of propagating plants asexually in aseptic conditions. The four main steps of micropropagation are establishment, multiplication, rooting and acclimatization. An additional step, regeneration, is needed for embryo tissue culture.

Tissue culture process for American chestnut somatic embryos


The Search for Blight Resistance-enhancing Genes

sampling canker from American chestnut treeTo enhance blight resistance in the American chestnut tree, we are studying several gene products, regulatory regions of genes to control expression (promoters), and the possibility of combining two or more genes (called stacking or pyramiding) for better and more sustainable resistance.

Search for resistance-enhancing genes


Demo of Blight Resistance

Controlled inoculations of chestnut stems with the blight fungus have confirmed that Darling trees with OxO have consistently smaller cankers than their non-transgenic relatives.

View time-lapse video of a small-stem blight resistance assay demonstrating the high level of blight resistance in two of our new transgenic American chestnut trees.

Small Stem Assay Method

Enhanced Blight Resistance in Transgenic American Chestnut

Enhanced Blight Resistance in Transgenic American Chestnut

Step by Step overview of the small stem assay method.a petri dish, marker, bottles and a sapling

1. Gather your supplies including an actively growing culture of Cryphonectria parasitica (the blight fungus). In these videos we used strain EP155.




making a sapling2. Make two marks with a felt pen 5 mm apart.





marking a sapling3. Carefully cut a consistent, vertical wound between the marks.





taking a sample from a petri dish4. Using a #2 cork borer, cut mycelial plug from the leading edge of the fungal colony.




5. Place mycelial side up on pre stretched parafilm.     sample in a petri dish




6. Press the mycelial plug against the wound making sure it stays in place.

tying a sapling





7. Wrap the parafilm tight to prevent drying of the plug and wait 7 to 21 days for results.                   

                                                          sapling in a pot