Gene editing can make 'chocolate trees' disease resistant

Scientists are using a powerful gene-editing tool to grow cacao trees that are more resistant to diseases, an advance that could help boost the production of chocolates all over the world.

The cacao tree, which grows in tropical regions, produces the cocoa beans that are the raw material of chocolate.

Reliable productivity from cacao plants is essential to the multibillion-dollar chocolate industry, the economies of producing countries and the livelihoods of millions of smallholder cacao farmers.

However, each year, several plant diseases severely limit global production, with 20-30 per cent of cocoa pods destroyed preharvest, said Andrew Fister, postdoctoral scholar from Pennsylvania State University in the US.

"In West Africa, severe outbreaks of fungal diseases can destroy all cacao fruit on a single farm," said Fister.

"Because diseases are a persistent problem for cacao, improving disease resistance has been a priority for researchers. But development of disease-resistant varieties has been slowed by the need for sources of genetic resistance and the long generation time of cacao trees," she said.

The study, published in the journal Frontiers in Plant Science, is the first to demonstrate the feasibility of using CRISPR technology to improve Theobroma cacao.

CRISPR stands for clustered regularly interspaced short palindromic repeats. It is a way to modify an organism's genome by precisely delivering a DNA-cutting enzyme, Cas9, to a targeted region of DNA.

The resulting change can delete or replace specific DNA pieces, thereby promoting or disabling certain traits.

Previous work in cacao identified a gene, known as TcNPR3, that suppresses the plant's disease response. The researchers hypothesised that using CRISPR-Cas9 to knock out this gene would result in enhanced disease resistance.

To test their hypothesis, they used Agrobacterium - a plant pathogen modified to remove its ability to cause disease - to introduce CRISPR-Cas9 components into detached cacao leaves. Subsequent analysis of treated tissue found deletions in 27 percent of TcNPR3 copies.

When infected with Phytopthera tropicalis, a naturally occurring pathogen of cacao and other plants, the treated leaves showed greater resistance to the disease.

The results suggested that the mutation of only a fraction of the copies of the targeted gene may be sufficient to trigger downstream processes, resulting in systemic disease resistance in the plant.

The researchers also created CRISPR gene-edited cacao embryos, which they will grow into mature trees to test the effectiveness of this approach at a whole-plant level.

This research builds on more than 30 years of biotechnology research aimed at building a better cacao tree, according to Mark Guiltinan, professor at Penn State.

"Our lab has developed several tools for the improvement of cacao, and CRISPR is just one more tool," he said.

"But compared to conventional breeding and other techniques, CRISPR speeds up the process and is much more precise. It's amazingly efficient in targeting the DNA you want, and so far, we haven't detected any off-target effects," Guiltinan said.

In addition to providing a new tool to accelerate breeding, CRISPR-Cas9 technology can help deliver insights into basic biology by offering a method to efficiently assess gene function, researchers said.

"With CRISPR, we can quickly 'break' a gene and see what happens to the plant," Guiltinan explained.