Increasing the efficiency of gene editing is especially important in wheat, which is particularly difficult to modify. This is because wheat is hexaploid, meaning it has 6 sets of DNA, increasing the amount of modification required compared to the two chromosomes in diploid organisms. Through traditional breeding, this is done through manual sorting over several generations, but it’s time-consuming and lacks precision. Researchers at the Innovative Genomics Institute sought out a solution to this problem by using new methods with CRISPR/Cas9 to improve transformation efficiency in the staple crop.

The most popular method of plant gene editing employs Agrobacterium, but some species are more difficult than others to transform. When Agrobacterium-mediated transformation fails, other methods of gene transfer can reach recalcitrant species. For example, particle bombardment entails the coating of tiny gold particles with DNA of interest, which are shot into plant cells to incorporate the foreign DNA. Using a gene gun, CRISPR/Cas9 can be injected straight into the nucleus without needing to use a vector like bacteria. Researchers suspected this was just the right tool for wheat, considering its tricky history.

The basic experimental structure was as follows: immature wheat embryos were bombarded with gold particles of various sizes and pressures coated with DNA using a particle gun. After two rounds of bombardment, the immature embryos went through different stages of tissue culture, including three rounds of selection under an antibiotic that only transformed plants had resistance to. Once full plantlets formed, they and their progeny were sampled for genetic information and observed for their phenotype. Using this biolistic technique and changing the variables involved in transformation, the research team set out to find the best way to genetically modify wheat.

To see what has been successfully transformed, the phytoene desaturase (PDS) gene, which is related to plant color, was involved in the transformation. PDS regulates the production of a pigment called β-carotene, so when it is knocked out by Cas9, plants with the edited genotype appear white. However, this had yet to be achieved before in wheat, which the researchers suspect wheat’s hexaploid genome to be responsible for, and that all six chromosomes containing the genes would need to be edited.

Although bombardment pressure did not have a significant impact on overall transformation efficiency, it was observed that the highest pressure was best for the largest particle size, and the lowest was best for the 0.4 μm and 0.6 μm particles. Of the three sizes tested (0.4, 0.6 and 1.0 μm), the middle size of 0.6 μm was found to outperform the others with an efficiency of 22.6%, much greater than the 10.7% and 9.0% seen in 0.4 μm and 1.0 μm respectively. Researchers noted that the same amount of gold was used in each trial, so the larger the particle, the fewer particles were expelled, possibly explaining the lower rates of the 1.0 μm particles. However, a smaller surface area includes less DNA, offering an explanation for the lower rates from the 0.4 μm particles.

In addition to biolistic conditions, transformation efficiency with CRISPR/Cas9 varies by other factors such as target species, DNA construct elements, and temperature. Adjusting temperatures has proven important in mammalian cell transformations, so these researchers tried applying the same logic to this plant transformation. Since the CRISPR/Cas9 being used was discovered in a bacterium that thrives most at 40 °C, which is hotter than typical plant cell cultures, the plants were subjected to a series of temperatures higher than normal. Increasing culture temperature up to 37 °C resulted in higher transformation rates, but reduced mass of transformed tissue, likely due to the adverse effects of high temperature on the plant tissue. Trials under 26 °C, 30 °C, and 34 °C fared better, but the researchers discovered two albino plantlets in the group under 34 °C. This means the mutants had PDS knockouts in every chromosome, the first known time this was achieved without subsequent breeding. Due to CRISPR/Cas9’s origin in higher temperatures, the higher temperature of this trial is thought to be responsible for this highly effective transformation.

These advancements in wheat transformation show significant promise for edits in wheat, which could have big implications for agriculture as such a prominent crop. Even outside of wheat, these discoveries can be applied to other recalcitrant crops to broaden the library of plants with efficient transformation protocols.

Somehow I had been under the impression that biolistics were an outdated technology in gene editing, which is why I hadn’t covered them on my website until now. It wasn’t until reading this paper, which came from my lab at IGI, that I realized how wrong I was, and what a useful tool a gene gun really is.

Just like with Agrobacterium, it is really satisfying to gain insight to how all of these biological advancements are made. There’s still so much I don’t know about the physical and molecular process of transformation, so it’s really exciting to get insight into these discoveries. I thought I would have a full understanding of everything I do in the lab once I became involved in wet lab research, and although my comprehension has certainly grown, I have room to grow. Just because you use a microwave doesn’t mean you understand how it works.

I would be interested to understand the pros and cons of biolistics vs. Agrobacterium, and how one might outperform another in different target species. I also wonder what work might be done on wheat now that the barrier to entry has been cracked open.

Stay tuned to learn with me!

https://pmc.ncbi.nlm.nih.gov/articles/PMC9318839/