Plant Transformation Facility
When Agrobacterium tumefaciens, a soil-dwelling, plant-pathogenic bacterium, infects a wounded plant, it inserts some of its own DNA into the host plants’ cells. In nature, the inserted genes cause infected plants to grow tumors. Scientists discovered in the 1980s that they could alter A. tumefaciens, replacing the tumor-inducing genes with genes sourced from another species.
Researchers start by inserting DNA from the source species into A. tumefaciens. The altered A. tumefaciens then inserts DNA from the source species into the cells of the target species. The resulting plant is transgenic, having genes from a different species incorporated into its own genome. The method does not work with surgical precision—A. tumefaciens inserts DNA randomly into the target plant’s genome, and not every cell exposed to the bacterium will become transgenic. It is still, however, one of the most efficient methods for creating transgenic plants.
Rather than using A. tumefaciens as a standalone technique for transgenesis, the PTF usually combines it with CRISPR-Cas9. The PTF is currently using A. tumefaciens with CRISPR-Cas9 to develop a transformation and genome editing system effective for industrial hemp. The system will help Larry Smart, School of Integrative Plant Science, Horticulture, and other hemp researchers at Cornell to improve hemp cultivars for New York State.
CRISPR-Cas9 genome editing harnesses a naturally occurring immune mechanism from bacteria. Rather than introducing a transgene from one species to another, CRISPR-Cas9 works with a species’ own genetic material—a process called genome editing.
In nature, bacterial species build a catalog of RNA sequences, based on the DNA of viruses that they have encountered in the past. If a virus infects the bacteria and has DNA that pairs with the bacteria’s catalog, an enzyme called Cas9 detects the match and inactivates the virus by cutting the virus’s DNA. What makes CRISPR-Cas9 remarkable is its precision. The RNA sequence guides the Cas9 enzyme to cut viral DNA at the exact location indicated by the RNA. Researchers in the early 2010s discovered that they could modify this bacterial immune mechanism to make targeted cuts to the DNA of any organism. The organism’s natural DNA repair mechanisms step in, knitting the cleaved DNA back together; but these repair mechanisms are not 100 percent effective. So a percentage of the targeted cuts result in edits—changes to the original DNA sequence.
Susan McCouch’s lab, School of Integrative Plant Science, Plant Breeding and Genetics, is working with the PTF and collaborators from Colombia to edit varieties of Colombian rice to require less fertilizer. The PTF has deleted a gene in these varieties that causes phosphorus to accumulate in the plant’s seed. Without the gene, more phosphorus remains in the plant’s leaves, which are left in the field at harvest. The leaf-bound phosphorus returns to the soil, reducing the amount of fertilizer required for the next crop. This in turn minimizes fertilizer runoff. In addition to the positive environmental impact, the transformed rice has an additional benefit: more zinc and iron accumulate in the grain, making it more nutritious. Upcoming tests will determine whether deleting this gene in Colombian rice varieties has the same benefits as seen in previously published reports.
The biolistics particle delivery system, also known as the gene gun, is an alternate to A. tumefaciens for making transgenic plants. Researchers at Cornell’s Agricultural Experiment Station in Geneva, New York (now Cornell AgriTech) and the College of Engineering invented the original gene gun in the 1980s. The biolistics system literally shoots DNA into a plant’s cells through its cell walls, carried by tiny gold particles averaging one micron in diameter. Some of the DNA makes its way into target plant’s nuclei, where it is incorporated at random into the target species’ genome.
When the devastating ringspot virus struck Hawaii’s papaya fields in the 1990s, Dennis Gonsalves, School of Integrative Plant Science, Plant Pathology and Plant-Microbe Biology (now emeritus), used the biolistics particle delivery system to create a transgenic variety, Rainbow papaya, that is resistant to the virus. Farmers in Hawaii and around the world still rely on the Rainbow papaya.
The PTF has pioneered a genome editing system that combines biolistics with CRISPR-Cas9. Other CRISPR-Cas9 methods necessitate a transgenic intermediate. These are plants that are transgenic for the two key components: the Cas9 enzyme and the RNA guide. Once the desired edit is achieved, the CRISPR-Cas9 transgenes are removed. Some countries regulate transgenic techniques more stringently than genome editing, and the PTF sought a technique that might comply with more restrictive regulatory spaces. By using the biolistics particle delivery system to introduce the CRISPR-Cas9 components, the PTF avoids the transgenic intermediate. The combined CRISPR-biolistics method also makes it possible to edit clonally propagated plants like bananas, apples, and grapes.
Growing a mature plant from a few undifferentiated cells is a delicate process. Each species, and even specific genotypes within a species, have unique nutritional demands. Technicians at the PTF must formulate customized growth media with the perfect consistency, acidity, nutrients, and hormones for each genotype researchers are working with.
Growth media play a crucial role in advancing tissue cultures from a few cells to a mature plant. To get undifferentiated cells to form shoots and then roots, researchers alter the levels of two hormones in the growth medium. High levels of both promote growth of undifferentiated tissue (top left); high levels of one and low of the other cause cells to differentiate, forming shoots (top right); finally, inversing the levels sparks the growth of roots (bottom left). To address high levels of oxidation produced by some species, technicians sometimes add charcoal to the medium, as seen here for hemp (bottom right).
Two growth rooms at the PTF provide the environmental conditions necessary for cultivating plant starts from undifferentiated cells. Petri dishes with plants at various stages of development line the shelves beneath growth lights. The PTF maintains one growth room at 28°C for rice and another at 25°C for most other species, including wheat, apple, grape, and industrial hemp.
Once plantlets are ready to grow in soil, the PTF can transfer them to greenhouses or growth chambers. The PTF has space in the Guterman greenhouses for growing maize and wheat. Growth chambers in the basement of Weill Hall are much like indoor greenhouses. Researchers can control almost every aspect of the environment, including the lighting, time of day, and temperature.
The PTF grows several rice varieties year-round in order to have a constant supply of immature rice seeds. In the growth chamber pictured here, the PTF is growing Carolina Gold, a variety of rice descended from the first rice grown in North America. Researchers believe Carolina Gold is derived from rice brought to North America in the seventeenth or eighteenth centuries through the slave trade. Adam Bogdanove’s lab, School of Integrative Plant Science, Plant Pathology and Plant-Microbe Biology, is identifying a gene in Carolina Gold that is crucial to disease resistance, research that prompted the PTF to develop a transformation protocol for the variety.
The Plant Transformation Facility (PTF) helps improve plant varieties by applying cutting-edge biotechnology to economically important crops such as, rice, corn, wheat, apple, and industrial hemp. Urgent agricultural challenges—drought, disease, and environmental impact and sustainability—motivate much of the research at the PTF. With a growing world population and climate change, plant biotechnology can help scientists increase yields and improve the food supply. The facility has a global reach with a particular commitment to New York State crops.
PTF staff typically begin with a small amount of plant tissue—often an embryo, seed, or leaflet—called an explant. They then alter the genetic makeup of individual cells in the explant, using transgenesis, genome editing, or a combination of both. Transgenic methods such as Agrobacterium tumefaciens and biolistics introduce genes from one species into another. Genome editing systems, CRISPR-Cas9 for example, delete and repair genes by making precision cuts in a cell’s DNA. From altered cells in the explant, researchers grow an entire plant that exhibits the desired traits. Plants have an amazing characteristic—unlike animals, every cell has the potential to act like a stem cell and give rise to a complete plant.
“We are at a really interesting point in time with technology that’s available to improve plants and to study plants. But there are not a lot of people out there who have the skills and knowledge to do it,” explains the director of the PTF Matthew Willmann ’99. “It also takes a lot of time to work all these things out and to do them, so this facility is critical to researchers on campus who want to use the technology but not do it themselves.”
“We’re working on a variety of crops that are vital to New York State agriculture,” Willmann adds. “For example, we are collaborating with Mark Sorrells, School of Integrative Plant Science, Plant Breeding and Genetics, to use CRISPR-Cas9 to help increase yield in his wheat breeding program and establishing grape transformation to help Jason Londo, School of Integrative Plant Science, Horticulture, study genes involved in cold tolerance. We are also working on some that are not vital to New York State, like rice, but that are vital to global hunger. And so we have this mix of helping the needs of our farmers and consumers here and helping the needs of people around the world.”
The PTF provides research services for a fee. It also trains students, postdocs, and visiting scientists in the use of these technologies.