Robustness is a characteristic that is highly desired for all plants that we grow, with the term covering the gamut of stress resistances and ability to defend oneself from pathogenic attacks. At the same time, however, agricultural breeders desire to bring in wild relatives of plants to provide a greater diversity of traits and, indeed, some enhanced robustness of their own. The downside to this is that said resistances are often highly focused only on the environment, stresses, and very specific pathogens that the wild relative encountered in its home region. So even if we are able to cross in useful traits, this usually results in other weaknesses being exposed and an extensive amount of cross-breeding is often necessary to split out the traits we want to keep from the ones we absolutely want to discard. 

Addressing Genome Duplication

That conflict is made even more difficult for the plant species that are self-incompatible and that have other incompatibility issues with cross-breeding as well. The amount of time required to drag out desirable traits in such a situation is a massive undertaking, to say the least. But gene modification technologies and CRISPR in particular have allowed for alternatives. Specifically, by using focused cutting to induce polyploidy, otherwise known as the duplication of genomes. Such events are actually rather common in plants and different tissues in the same plant can have different ploidy levels as well, with some cells having three copies of the genome, others having many more. 

An increase in ploidy level directly correlates to enhanced robustness, as there are more copies of genes that have the potential to mutate without removing other copies of the same gene, thus allowing for new traits to form much easier. Therefore, cultivars of crops that we make into tetraploids, or four copies, are that much more capable of dealing with the issues of climate change we’ll be facing in the future. And which is why past researchers have already accomplished this with wild rice just last year. Now, scientists at Academia Sinica in Taiwan have come together to do the same with the wild tomato, Solanum peruvianum, that represents the largest reservoir of disease resistance genes in the genus, and so would benefit from increasing its ploidy and allow for potential introduction of those genes into the other tomato cultivars we grow for food. 

Regenerating the Protoplast

To accomplish this and to also better investigate the effects that CRISPR has on polyploid genomes, particularly the variance across cells, the research team decided to find a way to employ the protoplast regeneration system to make it work with the gene editing tool. A protoplast in this context is a plant cell or the cells of an entire tissue that has had their cell walls removed. This then allows for foreign DNA and things like the CRISPR complex to be introduced into the cells without having to use an intermediary such as Agrobacterium. 

The benefits of this system is that it has a lower likelihood of producing chimeric cells, ie cells that have genomes that no longer match the rest of the tissues due to the modification, and also can be done with germ cells that pass on the changes to the next generation, unlike with Agrobacterium where often flowers and developing seeds have to be exposed in order to ensure the genetic changes have a likelihood of being passed on. Additionally, protoplast regeneration gets around the issue of self-incompatible species that were mentioned prior. The main downside of the process, however, is that it requires you to do protoplast regeneration in the first place. And that is, unfortunately, a major hurdle due to just how complicated doing so successfully is in itself, along with potential enhanced mutations occurring in regenerated tissues. 

A System For Trait Extraction

Therefore, the team worked on establishing a protoplast regeneration protocol using the wild tomato species so that it could be much more easily performed alongside CRISPR gene editing and controlled in a more stable manner. The genes targeted with the latter for knockout were several involved in RNA silencing and other pathogen defensive activities and regulatory pathways, due to this allowing for an easy check on phenotypic impact for the regenerated plants. 

In vitro shoots were regenerated from original plant tissue with a focus on tetraploid cells and various enhancements to the chemicals and environment for regeneration to occur were optimized. In total, they were able to create tetraploid versions of the wild tomato with CRISPR edits, but no transgenic material left in the cells and an overall efficiency of 60% in editing the tissues’ cells. The genomes were also found to be stable after the protoplast regeneration process and, as expected, more highly susceptible to particular viral pathogens due to successfully incapacitating several genes involved in viral defense. 

This protocol can serve as a basis for future combinations of protoplast regeneration and CRISPR editing and induced polyploidy events to create cultivars with higher copy amounts of the genome. All without having to involve direct inclusion of transgenes or having the CRISPR complex remain in subsequent tissues. The scientists hope to continue their research now that they’ve established this method and begin in earnest the experiments needed to begin extracting the useful traits that can be combined with domesticated cultivars and benefit tomato farming as a whole. 

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Photo CCs: Unripe tomatoes from Wikimedia Commons

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