Once a staple in hardwood forests stretching from Maine to Georgia and Mississippi, the American chestnut's domination effectively came to an end in the early 20th century when a massive blight wiped out billions of mature trees.

Now, researchers from the State University of New York College of Environmental Science and Forestry are working to restore the American chestnut's place in U.S. forests. The team's latest work provides new recommendations for increasing the stock of blight-resistant trees, using genetic engineering.

A fungus scientifically known as Cryphonectria parasitica is said to be the primary agent of the chestnut blight. The idea is, after the initial infection of wounded bark, the genetically-modified trees will be able to stop the fungus from spreading further behind a layer of plant tissue.

"The addition of one gene, oxalate oxidase, can protect the American chestnut by breaking down the oxalic acid secreted by the fungus," said Allison Oakes, corresponding author of the study.

The problem is hundreds of American chestnut trees are needed for field trials and eventual restoration plantings, but it is difficult to make hardwood trees produce roots through micropropagation.

"The presence of roots and living shoot tips precede successful acclimatization of tissue culture-produced American chestnut plantlets," researchers explained.

To improve the post-rooting stage of American chestnut propagation protocol, researchers created four experiments to test vessel type, concentrations of humic acid, activated charcoal and use of a vermiculite substrate.

Their study revealed that the presence of activated charcoal in the post-rooting stage significantly increased the percentage of rooted plantlets. Furthermore, increasing concentrations of activated charcoal had a significant impact on number of length of roots.

"High concentrations of humic acid combined with activated charcoal had excellent and significantly higher root presence than all the treatments without activated charcoal," the authors wrote.

Overall, researchers found an "overarching trend" of activated charcoal enhancing root growth but slightly decreasing shoot tip survival.

"Our findings have broad implications for tissue culture and genetic engineering of target hardwood species," Oakes added. "The combination of humic acid and activated charcoal in rooting medium more than doubled the number of roots produced by micropropagated shoots than either compound alone. Vermiculite substrate may be an alternative with additional research and methods development."

Researchers believe their findings have implications for other difficult-to-root hardwood trees in transgenic programs, such as American butternut, white oak and black walnut.

Their study was recently published in the journal HortScience.