An easier way to engineer plants

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Using nanoparticles to deliver genes into plant chloroplasts

A new genetic tool to facilitate plant design
Their technique, which uses nanoparticles to deliver genes in plant cell chloroplasts, works with many plant species, including spinach and other vegetables.

This new strategy could help plant biologists to overcome the difficulties associated with genetic modification of plants, a process that is normally complex and time-consuming, and must be adapted to specific plant species undergoing modification. "This is a universal mechanism that works for all plant species," explains Michael Strano, Professor of Chemical Engineering at Carbon P. Dubbs at MIT, about this new method.

Strano and Nam-Hai Chua, Vice President of the Temasek Life Sciences Laboratory at the National University of Singapore and Professor Emeritus at Rockefeller University, are the main authors of this study, which appears in Nature Nanotechnology.

"This is an important first step towards the transformation of chloroplasts," says Chua. "This technique can be used for the rapid screening of candidate genes for chloroplast expression in a wide variety of crops."

Targeting chloroplasts
A few years ago, Strano and his colleagues discovered that by adjusting the size and electrical charge of nanoparticles, they could design nanoparticles that could penetrate plant cell membranes. This mechanism, called lipid exchange envelope penetration (LEEP), allowed them to create plants that shone by incorporating nanoparticles containing luciferase, a light-emitting protein, into their leaves.

As soon as the MIT team announced that it had used LEEP to introduce nanoparticles into plants, plant biologists began to ask whether it could be used to genetically modify plants and, more specifically, to introduce genes into chloroplasts. Plant cells contain dozens of chloroplasts. Therefore, the indication of genes by chloroplasts (instead of the nucleus) could be a way to generate much larger quantities of a protein.

Chloroplast, better known as the site of photosynthesis, contains about 80 genes encoding the proteins needed for photosynthesis. The chloroplast also has its own ribosomes, which allows it to assemble proteins within the chloroplast. Until now, it has been very difficult for scientists to introduce genes into the chloroplast: the only existing technique was to use a high-pressure "gene gun" to force the genes into the cells, which could damage the plant.

Carbon nanotubes
Using their new strategy, the MIT team created nanoparticles made of carbon nanotubes wrapped in chitosan, a natural sugar. DNA, which is negatively charged, binds freely to positively charged carbon nanotubes. To introduce the nanoparticles into the leaves of the plants, the researchers applied a needle-free syringe filled with the particulate solution to the underside of the leaf surface. The particles then enter the leaf through tiny pores called stomata, which normally control the evaporation of water.

Once inside the leaf, the nanoparticles pass through the plant's cell wall, cell membranes and then the chloroplast's double membranes. Once the particles have entered the chloroplast, its slightly less acidic environment causes the release of DNA by nanoparticles. Once released, DNA can be translated into proteins.

In this study, the researchers provided a gene for the yellow fluorescent protein, allowing them to easily visualize which cells in the plant expressed the protein. They found that about 47% of plant cells produce the protein, but they think it could be increased if they could release more particles.

"This approach reported here certainly opens up new avenues of research into the introduction of chloroplast selective genes for transgenic expression in plants, as shown here by several species. "said Sanjay Swarup, Associate Professor of Biological Sciences at the National University of Singapore, who did not participate in this research.

More resistant plants
A major advantage of this approach is that it can be used on many plant species. In this study, researchers tested it on spinach, watercress, tobacco, rocket and arabidopsis thaliana, a type of plant commonly used for research. They also showed that this technique is not limited to carbon nanotubes and could possibly be extended to other types of nanomaterials.

The researchers hope that this new tool will make it easier for plant biologists to integrate various characteristics into vegetables and crops. For example, agricultural researchers in Singapore and elsewhere are interested in creating leafy vegetables and crops that can grow at higher densities for urban agriculture.

Other possibilities include the creation of drought-resistant crops; industrial crops such as bananas, citrus fruits and coffee must be resistant to fungal infections, which threaten to eliminate them, as well as modifying rice so that it does not remove arsenic from groundwater.

Genes can be passed on to the next generation
Since the modified genes are carried only by chloroplasts, which are inherited by the parent plant, they can be transmitted to the next generation and cannot be transferred to other plant species.

"This is a big advantage, because if pollen has been genetically modified, it can spread to weeds and you can produce weeds that are resistant to herbicides and pesticides. Since chloroplast is transmitted by maternal route, it is not transmitted by pollen and the level of gene containment is higher," explains Lew.