"It's about combining nature with tech, and it's much less daunting then you think." --Jane Lalonde, CMO Zea
The general public tends to be skittish around genetically modified organisms. The idea of scientists toying with the elemental building blocks of life on earth is understandably unnerving to people who don’t spend every day immersed in the world of biotechnology. To us, it’s much less daunting because we see that genetic modification is not about creating unnatural mutant organisms; it’s about combining nature with technology to make the earth’s natural systems work better with us. The end result may not be entirely natural, but it is a process guided by nature. Many breakthroughs in modern medicine are thanks to scientists genetically modifying organisms to produce drugs or deliver gene therapies. So, for those of you interested in knowing how these alterations are achieved, here are five different methods that can be used to deliver genetic information into a plant.
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This method of gene transfer involves deliberately exposing a plant to a genetically modified Agrobacterium tumefaciens phytopathogen (plant virus). Scientists insert a gene into the virus, and then the virus delivers the genetic information into the plant cell where it becomes integrated into the plant’s genome. A practice called “agroinfiltration” is commonly used in which the Agrobacterium is injected into a small prick in the plant’s tissue using a needleless syringe.
Agrobacterium-mediated transfer is a common technique for introducing vaccine candidates into a plant because it causes relatively little harm to the host organism and doesn’t diminish the immunogenic properties of the vaccine. As one research article states, “Microbial genes when incorporated into the plant genome are transcribed into protein antigens of the target pathogen without affecting the plant and without losing their immunogenic property.” (Dhama et al., 2020). Vaccines that are made using this viral vector are those that target tuberculosis, dengue, avian flu, and Ebola viruses.
Sometimes referred to as the “gene gun method,” a biolistic approach to gene transfer involves bombarding plant cells at high speeds with nucleic-acid-coated metal particles (often gold or tungsten). Nucleic acids are the building blocks of all life on earth, fundamental to both DNA and RNA molecules. By covering microscopic metal particles with this genetic material and shooting them like bullets at the targeted plant cell or organelle using a pressurized gun, scientists are able to infiltrate (transfect) the genome and introduce a new genetic trait to the organism.
Since this method relies on bombardment, almost any tissue and cell type could be affected using a gene gun. As a non-viral vector, it doesn’t have to deliver the genetic information to a specific target in a specific way. As shown in the diagram above, whatever is in the dish below the screen is likely going to be hit by the particles. It’s a straightforward and effective method. The disadvantages are that, since it is a brute force technique, it is sometimes imprecise and messy. A specific target cannot be controlled. Also, the gold projectiles, microscopic as they may be, can get expensive. Biolistics are often used for agricultural products. Some vaccines produced using biolistics are those that protect against cholera, Lyme disease, anthrax, tetanus, plague, rotavirus, and canine parvovirus.
This gene delivery system is based on the scientific observation that cell membranes become transiently permeable when short electronic pulses are applied to them. Electropolation uses these external electronic pulses to briefly open up plant cell membranes, allowing for genetic information to be admitted. Since electroporation works on living cells, it has also been used to deliver drugs such as chemotherapy as well as dyes/tracers for research purposes. Electroporation has reportedly been very helpful in administering cancer treatments. “Electroporation is used to enhance drug diffusion and gene delivery into the cytosol. The combination of electroporation and cytotoxic drugs, electrochemotherapy (ECT), is used to treat metastatic tumor nodules located at the skin and subcutaneous tissue. The objective response rate following a single session of treatment exceeds 80%, with minimal toxicity for the patients.” (Cadossi et al., 2014)
Another non-viral method of gene transfer, sonication uses ultrasound vibrations to disrupt the cell membrane, allowing for large molecules to enter intact. Like electroporation, the idea is to create transient holes in the membrane where DNA/RNA can pass through. An advantage of sonication is that it reportedly causes minimal damage to the cell. Studies from as early as 1997 state that, “The effects of sonication on plasmid DNA were investigated and indicated that the structural integrity of plasmid DNA was unaffected by the sonication conditions employed.” (Wyber eta al., 1997) And luckily for us, the technology has only gotten better in the past 23 years.
This petroleum-derived polyether compound is used to induce DNA uptake in plants. Plant protoplasts are treated with PEG in the presence of divalent cations (i.e. calcium). Divalent cations are negatively charged ions that have a valence of two, meaning they can bond with two negatively charged anions. The PEG and the divalent cations work to destabilize the plant’s plasma membrane, causing it to become permeable to the DNA molecules. Once the PEG treatment has rendered the plant protoplast transient, naked DNA can enter the cell’s nucleus and integrate into the host genome.
One benefit of PEG gene transfer is in its potential ability to harmlessly facilitate the insertion of DNA into a cell. In certain species of fungi and bacteria specifically, there was no damage at all caused to the cell wall after PEG gene transfer. Polymeric carriers also “have many advantages as gene carriers such as low cytotoxicity, low immunogenicity, moderate transfection efficiency, no size-limit, low cost, and reproducibility.” (Minhyung Lee & Sung Wan Kim, 2005)
With all of these methods, the goal is to carefully infiltrate the plant’s cell membrane and deliver DNA/RNA into the nucleus or chloroplast where it becomes a part of the plant’s genetic makeup. As we’ve seen, the benefits to society – specifically in the field of biologic medicines – has been incalculable. And the potential of genetically modified plants to provide us with even better biologic drugs makes innovations in biotechnology a worthwhile pursuit in our eyes.
To recap, the viral route for gene transfer uses the natural delivery mechanisms of a pathogen to insert genes into a plant. The biolistic route forces the genetic information into the plant cell by firing projectiles into the cell using a gene gun. Electronic and sonic pulses can be used to open up the cell membrane and PEG can similarly be used to disrupt the membrane just enough to allow DNA in. Genetic modification is something to be careful with, to be sure. However, it can provide the world with some truly exciting and innovative products. Plant-made pharmaceuticals, for example, greatly benefit world health and they rely heavily on recombinant proteins and genetically modified organisms.
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