Two articles recently published in the International Journal of Molecular Sciences and the Plant Cell, Tissue and Organ Culture journals outline how CRISPR technology can advance the uses of the world's largest vegetable crop.
Both papers include the work done by Stephany Toinga, Ph.D., who was a graduate student in the lab of Keerti Rathore, Ph.D., AgriLife Research plant biotechnologist in the Texas A&M Institute for Plant Genomics and Biotechnology and Department of Soil and Crop Sciences. Also co-authoring both papers was Isabel Vales, Ph.D., an AgriLife Research potato breeder in the Texas A&M Department of Horticultural Sciences. Toinga is now a Texas A&M AgriLife Research postdoctoral associate with Vales.
"The information and knowledge we gained from these two studies will help us introduce other desirable traits in this very important crop," Rathore said. Developing potato cultivars with modified starch could open new opportunities, Toinga said. Potatoes with high amylopectin and low amylose, like the gene-edited Yukon Gold strain she described in the International Journal of Molecular Sciences, have industrial applications beyond traditional uses.
In contrast, potatoes with high amylose levels and low amylopectin would be desirable for human consumption, Vales said. The amylose acts like fiber and does not liberate glucose as easily as amylopectin, thus resulting in a lower glycemic index and making potatoes more acceptable for people with diabetes. CRISPR/Cas9 technology has expanded the toolset available to breeders, Vales said, and it represents a more direct, faster means to incorporate desired traits into popular commercial crop varieties. Conventional breeding is a lengthy process that can take 10-15 years. In addition, she said, due to the complex nature of the potato genome, generating new cultivars with the right complement of desirable traits is challenging for conventional breeding. Molecular breeding has enhanced breeding efficiencies, and gene-editing using the CRISPR/Cas9 technology adds another level of sophistication.
"We utilized the Agrobacterium method to deliver the CRISPR reagents into potatoes because it is reliable, efficient and least expensive compared to all other delivery methods," Rathore said.
In the first study, highlighted in the Plant Cell, Tissue and Organ Culture article, a potato line containing four copies of gfp, a jellyfish gene that allows a fluorescence-based visualization of the gene's activity, was targeted for mutation using the CRISPR/Cas9 system, Toinga said. In essence, this project provided an easy-to-see trait that enabled researchers to optimize the methodology.
"Loss of the characteristic green fluorescence and sequencing of the gfp gene following CRISPR treatment indicated that it is possible to disrupt all four copies of the gfp gene, thus confirming that it should be possible to mutate all four alleles of a native gene in the tetraploid potato," Rathore said.
Among the various potato cultivars evaluated in the first study, the Yukon Gold strain regenerated the best, and so it was used for the second study. In the second knockout study, described in the International Journal of Molecular Sciences, the native gene gbss in the tetraploid Yukon Gold strain was targeted to effectively eliminate amylose. The result was a potato with starch rich in amylopectin and low in amylose.
"One of the knockout events, T2-7, showed normal growth and yield characteristics but was completely devoid of amylose," Toinga said. That tuber starch, T2-7, could find industrial applications in the paper and textile sectors as adhesives/binders, bioplastics and ethanol industries. Tuber starch from this experimental strain, because of its freeze-thaw stability without the need for chemical modifications, should also be useful in producing frozen foods. Potatoes with amylopectin as the exclusive form of starch should also yield more ethanol for industrial use or to create alcoholic beverages.
As the next step for these studies, the T2-7 strain has been self-pollinated and crossed with the Yukon Gold strain donor and other potato clones to eliminate the transgenic elements.
(Source: Agriculture and Food News, ScienceDaily.

A depiction of the process for the elimination of amylose starch in a potato. Photo Credit: Texas A&M AgriLife graphic