The creation of transgenic plants and the use of gene modification technologies has been a marvel for agriculture and medicine, with the former seeing the ability to massively increase resistances to stress and higher production and yield possibilities. But, at the same time, even this sort of technology is slow and requires multiple generations of breeding to ensure the new traits are stable and can then be further backcrossed into high quality cultivars. All of this process requires an extensive amount of time and money to complete for even a single new transgenic trait to be able to go to market. 

So ways to increase the productivity and efficiency of the overall mechanism have been a constant research focus, along with looking into alternative options that could be just as impactful in controlling gene expression. One such option avoids direct genetic modification altogether and instead aims to silence selective genes within a living plant. As can be expected, this system isn’t simple and has had significantly lower efficiencies than what would be required to have it be used on a large scale. However, efforts to improve that have been ongoing and now researchers at Kyoto University and the RIKEN Center have teamed up to showcase what they’ve accomplished. 

How To Infiltrate

The use of bioactive molecules has shown the capability to infiltrate into plant leaves and into the cells themselves with genetic components like small interfering RNAs (siRNAs) being used to interfere with the expression of selected genes. To succeed in this infiltration, a nanocarrier delivery system must be used that binds with the bioactive molecules and allows for them to be carried into the plant tissues. Studies over the past five years have shown that it is possible to use such a system to go into the cytoplasmic space inside leaf cells and even be targeted at particular organelles, such as the chloroplast. 

This is done by using cell-penetrating peptides (CPPs) that can passively ferry the biomolecules through the cell wall and, combined with organelle-targeting peptides, can further take the biomolecules to the genomic material that is being targeted. The research team in today’s study wanted to broaden this capability and create a platform that would allow for nanocarrier genetic silencing to be done to multiple plants all at once in the form of a leaf spray. This would allow for both natural and synthesized CPPs to be used on particular plants on a larger scale. 

Making An Effective Spray

After first testing their sprays on the model organism Arabidopsis thaliana, they then included crops such as soybeans and tomatoes in order to determine which of the many CPPs available for use would be most efficient in infiltrating leaf tissues. Since it is entirely possible that the most efficient option would differ between plant species dependent on cell thickness and other variables. 

To present the effectiveness of the sprays they were working on, they used a transgenic Arabidopsis with a fluorescent yellow protein trait that then had that gene targeted by the spray for silencing and thus removing the yellow fluorescence. Also, to confirm that the carrier complexes were being delivered to the correct places in the cells and the total number of affected cells, they used a GUS reporter gene combined with the carrier so they could track the infiltration. And then used Agrobacteriam to conduct the transgenic inclusion. 

They found that their spray testing with yellow fluorescence resulted in around a 50% decrease in gene expression after application with the spray. Their tests on tomatoes, meanwhile, saw a much higher efficiency result at 70+%. This led to further tests using green fluorescent protein instead to see if the chloroplast genome could be affected instead of just the nuclear genome. The spray did succeed in reducing GFP expression and so gave a solid boost to the idea of having greater genomic silencing control over both the chloroplast and mitochondrial genomes. 

More To Do

Though how the spray is succeeding within these organelles remains poorly understood, as the existence of an RNA interference-based system has yet to be definitively confirmed in either. So how effective and efficient this would be on those non-nuclear genomes on a larger application isn’t known as of yet, nor is the effectiveness of the bioactive molecule spray on the mitochondrial genome in general. Further testing is required in that regard.

But even if these large platform nanocarrier sprays only work at relevant levels on nuclear genes, that is still a tremendous success in creating short term gene knockout systems in living plants. This can’t be used to actively add new traits as with other kinds of genetic modification tools, at least not yet, but the speed and versatility of being able to create changes in living plants without requiring multiple generations outstrips those downsides. 

Hopefully in the future, this method of altering plants can be integrated into all the systems we use and can assist in short term, immediate gene silencing changes that are required for responses to sudden environmental changes going on at the same time. It certainly seems like dealing with the uncertain future of climate change impacts would be benefited by having such a rapid response tool at hand.

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Image CCs: Grant Clark County Wisc soybeans from Wikimedia Commons

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