Researchers have developed a gene-editing system designed to overcome a major bottleneck in plant biotechnology by helping edited cells regenerate into whole plants more quickly and reliably.
The study, led by researchers at Texas A&M AgriLife Research, the University of Maryland and the U.S. Department of Agriculture, describes CRISPR-Combo, a system that allows scientists to edit a gene of interest while simultaneously activating the plant's own morphogenic genes. The findings were published in Nature Communications.
Morphogenic genes control how plant cells divide and develop into roots, shoots and eventually entire plants. Rather than inserting additional copies of these growth-promoting genes, CRISPR-Combo activates the plant's existing genes in their native locations using the same CRISPR technology used for the desired gene edit.
The approach could help improve gene-editing efforts in crops that are difficult to transform or regenerate, including perennial crops such as citrus, strawberry and poplar, as well as potatoes.
Regeneration is a significant challenge in developing gene-edited crops because scientists must be able to grow a complete plant from a small number of edited cells. The process can be slow or unreliable, and some plant species have proven difficult or impossible to regenerate using conventional laboratory techniques.
Perennial crops can present an additional challenge because they may take years to complete a single breeding cycle. Many high-value fruit and nut crops also have low transformation and regeneration efficiency.
The researchers first used a high-throughput hairy root system developed by AgriLife Research to screen potential morphogenic genes. The system promotes root growth on plant cuttings, allowing researchers to evaluate candidate genes without going through the lengthy process of regenerating an entire plant.
In potatoes, researchers screened 17 candidate genes and identified four that increased hairy root production. Three of those genes also improved shoot regeneration, increasing regeneration efficiency to 45% to 70%, compared with about 30% to 35% in control plants.
The team also screened 10 candidate genes in citrus, a crop that has historically been difficult to transform and regenerate. Five genes significantly increased hairy root formation. Under laboratory conditions, all five increased shoot regeneration efficiency to at least 80%, compared with less than 60% in control plants.
Researchers tested the approach further in wild strawberry and poplar by activating two morphogenic genes simultaneously.
In strawberry, activating specific combinations of genes reduced the time required to produce a fully regenerated, gene-edited plant by more than a month compared with standard methods.
In poplar, simultaneous activation of two genes allowed shoots to regenerate in less than a month without the use of external plant hormones normally required for tissue culture. Poplar lines with both genes activated also had the highest rates of edited cells and developed into taller, higher-biomass plants in greenhouse testing without obvious abnormalities.
The researchers said the approach could provide a more streamlined and scalable method for improving plant regeneration. Because CRISPR-Combo activates genes already present in the plant rather than relying on additional genetic material or external hormones, the system also could be used to identify morphogenic genes in other commercially important crops that are difficult to regenerate using conventional methods.
The research was supported primarily by the Foundation for Food and Agriculture Research's Genotype-Independent Regeneration of Fertile Plants program, with additional support from the U.S. National Science Foundation, USDA National Institute of Food and Agriculture, U.S. Department of Energy, Texas A&M AgriLife Institute for Advancing Health Through Agriculture and AgriLife Research's Insect Vector Disease program.
Source: Texas A&M, "New gene-editing tool speeds regeneration of hard-to-breed perennial crops"