Green Biotech: Methods of Genetic Engineering in Plants
Green Biotech: Methods of Genetic Engineering in Plants
by Tawni Bull
Today we are going to focus on my favorite branch of biotech, green biotech. Like I mentioned in the first blog of this series, green biotech encompasses the agriculture industry and how scientists and researchers are developing more sustainable production of crops and improving those crops through genetic engineering. There are many parts to this branch including crop modification for improved traits through transformation and tissue culture, bioremediation, and production of biofuels, bioenergy, and biofertilizers. My specific research focuses on improvement of crops through tissue culture and transformation. Therefore, today we are going to focus on different methods of genetic engineering in plants and methods used to accomplish these.
But first, let’s define a term: genetic engineering. This is the process of deliberately modifying the genetic material (genotype) of an organism with hopes to observe desirable characteristic (phenotype) changes. Genetic engineering can be achieved through inserting new fragments of DNA (gene delivery), deleting or editing existing DNA by targeting a specific sequence, and/or random mutagenesis with the use of chemicals, UV radiation, or transposons. Today we will only be discussing gene delivery, the methods by which we accomplish this, and its applications. If interested, this article discusses the background of CRISPR and base editing and here you can learn more about random mutagenesis for genetic engineering.
Now, let us talk about two main methods used for gene delivery: 1) vector-mediated gene transfer which includes Agrobacterium-mediated plant transformation and 2) direct (or vector less) DNA transfer such as particle bombardment.
Agrobacterium-mediated Transformation
Agrobacterium is a genus of bacteria that can cause the development of tumors in plants through a process known as horizontal gene transfer. There are multiple species within this genus, but we will only be focusing on one, Agrobacterium tumefaciens. A. tumefaciens is the most commonly studied species in this genus and is responsible for a disease, called crown gall disease, found in over 140 plants. The basis behind this disease is a unique process in which a segment of DNA, termed the transfer DNA or T-DNA, is transferred from the bacterium into the plant. The T-DNA will make its way into the nucleus of the plant, where it will become randomly integrated into the plant genome. Once integrated, the plant itself, will express the genes located within the T-DNA region causing prolific cell division which results in the formation of a tumor (Figure 1.).
In 1977, Zambryski et al. engineered the tumor-inducing (Ti) plasmid (the circular genetic material that contains the T-DNA fragment in A. tumefaciens) by replacing the natural T-DNA fragment with a different gene of interest (GOI) and demonstrated it was possible to transfer the engineered T-DNA into a plant cell. This was the first instance where scientists “hi-jacked” this natural process by cloning a GOI into the T-DNA region and transforming plant cells in a laboratory setting. Over 40 years later, we are still using this as a main method of genetic engineering plants and continue to find ways to improve the process. One example of a study using this method is the introduction of a cytochrome P450 gene into pineapple which allows the plant to monitor and degrade persistent organic pollutants from the environment (Ma et al. 2012). This has been used across many different species of plants, however, not all plants are susceptible to this type of infection and, therefore, cannot be transformed using A. tumefaciens.
Particle Bombardment
Due to some limitations of the Agrobacterium-mediated transformation methods, another form of transformation emerged in 1987. This method of plant transformation is known as particle bombardment. Initially termed “biolistics”, this approach bombards plant cells with DNA-coated particles, typically gold or tungsten, at very high speed (~1,000-2,000 feet/second) (Sanford et al. 1987). Once inside the plant cell, the particles release the DNA where it can be integrated into the genome. Figure 2 explains, in more detail, how this process works. Some other fun names for this process include the gene gun and bio-blaster. A recent study applied the particle bombardment process to develop a Alstroemeria, a common ornamental and house plant, that was resistant to Alstroemeria Mosaic Virus (Kim 2020). You can read more about this here.
You may be wondering what happens after we deliver a gene to a cell, and how does that cell turn into a full plant? We can accomplish this through a process known as tissue culture. This is an in vitro, aseptic technique used to develop plants from a single cell. When transforming a plant cell, we start with a piece of excised plant tissue called an explant. Get it? Excised plant equals explant. The type of explant can be from mature leaves, immature leaves (cotyledons), stem tissue, parts of the flower (anthers, ovaries, stigmas, etc.), and even roots. Once these tissues are excised and transformed, they are place on petri plates containing selective, nutrient media. One important point I did not mention earlier is that within the engineered T-DNA fragment or DNA fragment bombarded into a plant cell is a selectable marker gene. This can include genes that confer resistance to antibiotics such as kanamycin (nptII) or hygromycin (hpt), or resistance to herbicides such as glufosinate (bar). Adding the selecting agent (antibiotic or herbicide) to the media allows for the selection of transformed (or transgenic) plants. Also included in the media are plant growth hormones, auxin and cytokinin, which promotes prolific cell division and production of totipotent cells (like stem cells) that can develop into any differentiated cell form. The ratio of auxin to cytokinin will help push the differentiation of cells toward shoot (above ground part of the plant) growth or root growth. Typically, we start by developing a shoot in culture, then developing roots, and then transferring to soil where we can grow the transformed plant in a green house. Although, there are some cases where root development precedes shoot formation. Figure 3. Demonstrates this process and includes pictures of some of my research involving tissue culture in the Michelmore Lab.
Gene delivery has been used to study all aspects of plant biology including pathology, physiology, and anatomy. So much research, in fact, I would not be able to scratch the surface of what has been accomplished using this technique. A few examples include the development of plants for molecular pharming (remember from Red Meets Green Biotech) by delivering genes that encode for pharmaceuticals, development of crops with higher nutrient content (Golden Rice), plants that are resistant to diseases and herbicides, and plants that can make their own insecticides (Bt Corn). Like I mentioned earlier, green biotech has many moving parts, but a large part of it includes crop improvement for food security and more sustainable farming practices. We can use gene delivery to do just that, deliver GOI into a plant cells for the development of improved crops. Next time, we will continue talking about green biotech by discussing different types of genetically modified organisms (GMO/GM), GM products currently available, and address some common misconceptions about GMOs. Make sure not to miss this one, it is going to be interesting!
References
Kim, J. B. 2020. Production of transgenic Alstroemeria plants containing virus resistance genes via particle bombardment. Journal of Plant Biotechnology. 47:164-171.
Ma, J. et al. 2012. Effective Agrobacterium-mediated transformation of pineapple with CYP1A1 by kanamycin selection technique. African Journal of Biotechnology. 11 (10):2555-2562
Sanford, J., et al. (1987). Delivery of Substances into Cells and Tissues using a Particle Bombardment Process. Particulate Science and Technology. 5:27-37.
Zambryski - https://www.embopress.org/doi/abs/10.1002/j.1460-2075.1983.tb01715.x
Editor's Note
This is the fourth blog by Tawni Bull in a five-part series "The Biotechnology Rainbow", including: