New CRISPR tool speeds up regenerating gene-edited perennial crops

Editing a plant's DNA is not the hard part. Once a gene has been changed inside a handful of cells, scientists still have to coax those cells into growing into a complete, functioning plant, a process called regeneration. For many species, especially perennials such as citrus, strawberry, poplar and potatoes, that step can be slow, unreliable, or simply fail.

A team led by researchers at Texas A&M AgriLife Research, the University of Maryland and the U.S. Department of Agriculture has described a system meant to speed up that step. The findings were published in Nature Communications. The system, called CRISPR-Combo, lets scientists make their intended gene edit and switch on the plant's own regeneration machinery in the same step.

"Regeneration is one of the biggest roadblocks standing between a promising gene edit in the lab and a crop variety that's actually useful to growers," said Kranthi Mandadi, director of the Texas A&M AgriLife Research and Extension Center at Weslaco and a professor in the Texas A&M Department of Plant Pathology and Microbiology. He said the approach shows a plant's own genes can be coaxed to regenerate faster and more reliably, which could help perennial crops like citrus that have historically been difficult to work with in the lab.

Inside a plant genome sit "morphogenic" genes, which control how cells divide and develop into roots, shoots and eventually a whole new plant. Standard lab techniques for growing plants from edited cells often rely on adding external plant hormones or inserting extra copies of growth-promoting genes into the genome. CRISPR-Combo instead uses the same CRISPR editing tool that makes the intended gene change to switch on the plant's existing morphogenic genes, right where they already sit in the genome, rather than adding new genetic material.

To find out which of these growth genes were worth activating, the team first used a fast screening method called a "hairy root" system, developed by AgriLife Research. It induces root growth directly on plant cuttings, letting researchers test many candidate genes without going through the slower process of growing whole plants from scratch. Manikandan Ramasamy, an AgriLife Research associate research scientist at the Weslaco center, is the study's primary author.

In potatoes, the team screened 17 candidate genes and found four that boosted hairy root production; three of those also improved shoot regeneration, the process of a cutting sprouting new stems and leaves. That raised regeneration efficiency to between 45% and 70%, compared with about 30% to 35% in untreated controls, according to Ramasamy.

Citrus has long resisted standard lab transformation and regeneration methods. Among 10 candidate genes screened in citrus, five meaningfully boosted hairy root formation, and all five pushed shoot regeneration efficiency to 80% or higher, versus under 60% in controls.

In wild strawberry and poplar, the researchers activated two morphogenic genes at once instead of just one. That combination shortened the time needed to produce a fully regenerated, gene-edited strawberry plant by more than a month compared with standard methods. In poplar, the effect was larger still: shoots regenerated in less than a month, and did so without any added external plant hormones, which tissue culture normally requires. The poplar lines with both genes activated also had the highest rates of edited cells and grew into taller, higher-biomass plants in the greenhouse, with no obvious abnormalities.

The difficulty of regenerating perennial and recalcitrant crops is a recognized problem in plant biotechnology more broadly. A review of citrus, coffee and sugarcane gene editing notes that CRISPR delivery and regeneration success depend heavily on the crop's genotype and the specific target gene, and that the choice of delivery method directly affects editing rates. Another review of CRISPR/Cas9 in plants describes existing delivery methods as inconsistent and inefficient for transforming whole plants, a gap the review says needs further research. Separate work on nodal culture, a tissue-culture technique using immature stem sections, has also been developed specifically to address regeneration failures in recalcitrant horticultural crops such as mango, mangosteen and coffee.

The AgriLife-led team believes CRISPR-Combo's approach, of screening for and activating a plant's own morphogenic genes rather than adding external ones, can be adapted to other commercially important crops that respond poorly to typical regeneration methods. The research was supported primarily by the Foundation for Food and Agriculture Research's Genotype-Independent Regeneration of Fertile Plants program, with additional funding from the National Science Foundation, the USDA National Institute of Food and Agriculture, the Department of Energy, and Texas A&M AgriLife programs.