ESF submitted a revised petition based on extensive feedback from regulators.

A labelling error was discovered. This mistake occurred between two Darling varieties(54 and 58) and that were developed at the same time.

Extensive testing has been done on Darling variety. The difference between Darling 54 and Darling 58 is the location of the new transgenes; the OxO gene is the same but located on a different chromosome. Data ESF has already provided to regulatory agencies confirms safety and characteristics of D54.

SUNY-ESF scientists reported that a different promoter, win3.12 from the eastern cottonwood, allows the expression levels of OxO to remain low in basal conditions, but increase under wound or infection. In laboratory bioassays, win3.12-OxO lines showed elevated disease tolerance similar to that exhibited by blight-resistant Chinese chestnut.^[20[1]^]

On November 10th, 2022, the U.S. Department of Agriculture published its draft Environmental Impact Statement and Plant Pest Risk Assessment. These statements found no reason to reject ESF’s application. 

SUNY ESF reported Oxo transgenic events passed onto viable progeny. ^{[11] [16]}

The Darling 58 (SX58) line was produced before 2016. Darling 58 is one of the transgenic events that produces the highest amount of OxO. ^[10]

Initial experiments to introduce wheat OxO into American chestnuts using different promoters to drive gene expression. Infection experiments on cut leaves show that blight lesion sizes can be reduced to around or below the level of the blight-resistant Chinese chestnut. ^[14][15]

The team began working with oxalate oxidase, OxO, which chemically inactivates the oxalate toxin, thereby protecting the trees from harmful effects of blight. OxO enzymes are widespread in the plant kingdom and other organisms. They are found in both food plants and wild organisms, including all grains, beets, spinach, switchgrass, mosses, and many types of bacteria and fungi. OxO does not directly harm the blight fungus; this enzyme simply breaks down oxalate, which reduces the pressure on the blight fungus to evolve resistance. This gene, when added to the 40,000 genes that comprises the American chestnut tree genome, detoxifies the fungus acid that would kill the tree.

ESF Professors William Powell and Charles Maynard began experiments to use new genetic techniques to provide substantial tolerance to Cryphonectria parasitica in American chestnut trees. The goal: to restore to our eastern forests American chestnut trees that can withstand the blight through transgenic biotechnology. Nothing like this had ever been done. Dr. Powell said “We basically had to build the boat before we went fishing“.