Two strategies have been developed by ICRISAT researchers to keep groundnuts nearly free of aflatoxin. In the first strategy, they have prevented fungal infection. And in the second strategy, they have prevented the fungus from producing the toxin even if it infects the groundnuts. The team led by Dr. Pooja Bhatnagar-Mathur intends to combine the two traits in a single variety to confer double protection to groundnut plants.
Researchers at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) in Hyderabad have developed dual strategies to keep groundnuts almost free of aflatoxin — a toxin produced by the fungi Aspergillus flavus and Aspergillus parasiticus — contamination. While one strategy prevents groundnuts from being infected by the fungus thereby preventing the toxins from being produced, the other strategy prevents the fungus from producing the toxin even if groundnuts somehow get infected with the fungus.
Genetic engineering approaches were used for inserting two alfalfa genes into groundnut plants to enhance immunity against fungal infection and growth. Preventing aflatoxin production even in case of any infection was achieved through a plant-induced gene silencing technique.
While both strategies showed promising results, the ultimate goal is to combine the two traits into a single variety to offer double protection so that groundnuts do not accumulate any aflatoxin or the amount of toxin is well within permissible limits at or after harvest.
Towards dual protection
“It is a proof-of-concept study. We have individually tested each of the two mechanisms and it is a matter of using conventional plant breeding approaches to develop a variety that has both the traits in place,” says Kiran K. Sharma from ICRISAT. “Only one of the two alfalfa genes and two RNA molecules studied may be needed to produce a variety.”
The researchers plan to start field trials early next year. “It will take one-two years to breed the two traits into a single variety and another about three years to conduct biosafety trials followed by the development of regionally adapted groundnut varieties. So, if everything goes to plan and gets approved by the Genetic Engineering Appraisal Committee (GEAC), farmers will have a groundnut variety that is near-immune to aflatoxin contamination in five to seven years,” says Dr. Pooja Bhatnagar-Mathur, Theme Leader – Cell, Molecular Biology and Genetic Engineering at ICRISAT and who led the team of researchers.
Nearly two billion people in sub-Saharan Africa and south Asia consume aﬂatoxin-contaminated agricultural commodities at levels far above the European limit of 4 ppb. Aflatoxin is a carcinogen and upon consumption does not get flushed out of the system. It instead gets bioaccumulated even leading to liver cirrhosis/cancer. The toxin is not visible and is tasteless even at very high concentration of 1,000 ppb making it difficult for consumers to avoid eating contaminated food items. In India, groundnut is one of common food items that show aflatoxin contamination.
How the groundnuts were made toxin-free
“We selected two specific genes from alfalfa and inserted them into groundnut plants to enhance the immunity against fungal infection and growth. Groundnuts showed very little fungal infection and negligible aflatoxin contamination,” says Dr. Bhatnagar-Mathur. “We choose alfalfa as it is a legume like groundnut.”
To further prevent toxin production even if groundnuts get infected with the fungus, the researchers designed two small RNA molecules that silence the fungal genes which produce aflatoxin. “When the fungus and plant come in contact with each other the small RNA molecules from the plant enter the fungus and prevent it from producing aflatoxin,” she says.
Though there are over 20 genes involved in aflatoxin biosynthesis, the team targeted only two genes to prevent toxin production. “We found that by targeting these two genes, the expression of five other genes in the alfatoxin biosynthetic pathway was affected,” says Sharma who is the first author of the paper published in Plant Biotechnology Journal. The research was done in collaboration with the U.S. Department of Agriculture, Louisiana State University and Donald Danforth Plant Science Center in the U.S.
Toxin less than permissible limits
About 40 hours after infection with Aspergillus, six lines with alfalfa genes showed less than 1 part per billion (ppb) of toxin and another five lines showed 1-4 ppb compared with over 3,000 ppb in groundnuts that did not have these genes. Similarly, six lines carrying the RNA molecules, the toxin present was less than 1 ppb and two other lines showed 1-4 ppb of toxin. “It is much lower than the Indian and U.S. safety limit of 20 ppb and meets even the stringent European safety limit of 4 ppb,” Dr. Bhatnagar-Mathur says.