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Though it may not be obvious from the piles of oranges available in dining halls and grocery stores, the USU.S. citrus industry is facing unprecedented threats from diseases that drastically reduce fruit yield. For example, huánglóngbìng, or citrus greening disease, has caused a seventy-five percent decrease in Florida’s citrus production. Unfortunately, like many other citrus diseases, there is no rapid, effective cure.
To address this challenge, scientists hope to genetically engineer disease-resistant citrus. This often involves using the bacteria Agrobacterium to deliver CRISPR/Cas9 gene-editing technology into the plant—a process called transformation. Plant cells are then isolated and incubated in plates or tubes, a process called in vitro tissue culture. Though widely used, this technique is laborious and costly, as many species are recalcitrant, or uncooperativeresistant, to tissue culture methods.
In their recent study, Yale postdoctoral associate Archana Khadgi and Yale Department of Mmolecular, Ccellular, and Ddevelopmental Bbiology professor Vivian Irish optimized a transformation protocol that is in planta, meaning that it does not require tissue culture. They tested various Agrobacterium application methods to determine which resulted in the best regeneration rate, or the percent of plants that regenerate their shoots after Agrobacterium inoculation. Furthermore, they studied the transformation rates, or percent of plants successfully gene-edited by CRISPR/Cas9.
Out of the six methods tested, the apical bud cut, growth, wounds on axillary meristems (AGWC) method “AGWC,” which stands for “apical bud cut, growth, wounds on axillary meristems,” resulted in the highest regeneration and transformation rates. Via the AGWC methodIn this method, the citrus seedling’s apical bud was cut and left for three days to grow its axillary meristems, where the plant grows secondary structures like branches. It was then “micro-wounded” by thin needles and covered with a cotton ball saturated with Agrobacterium. Optimal humidity, dark incubation methods, and seedling age were also tested. It was found that younger seedlings that were four to six weeks old and incubated in the dark with ninety-five percent relative humidity had the best regeneration and survival rates.
Seven gene-edited lines of the commercially used 8A Lisbon lemon, which is highly recalcitrant to conventional tissue culture approaches, were obtained using this new approach. The researchers observed that the regeneration rate across all cultivars—specially bred plant varieties— significantly increased, growing to over ninety-five percent in the Lisbon lemon. Moreover, most plants had high knockout scores of over ninety percent, a measure of successful gene edits. Such efficiency entails fewer resources, greater output, and successful gene editing for future plant genetic engineering. “The whole process […] was super rapid; it took less than three months. But the same in in vitro can take over a year. So timewise, it is very efficient. With this [protocol], [the plants] are growing directly in soil, [and] you just wait for them to grow, so it’s very labor-efficient as well,” Khadgi said.
This in planta transformation method allows more efficient and effective genetic engineering of commercial citrus crops for disease resistance, an invaluable tool for farmers and the US agricultural industry. “The most important thing right now is having a protocol that would work on recalcitrant varieties. […] We can now adapt this protocol in several other citrus cultivars that were very difficult to transform,” Khadgi said. Thanks to researchers like Khadgi and Irish, we can hope to continue seeing an abundance of citrus in our stores.