Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
For much of the world, rice is a key source of sustenance. For Bing Yang, it is a research subject. Botanists continue to embrace the latest in molecular tools to advance and better understand their evolving transgenic creations. Here we cover some of the latest techniques and strategies that leading plant researchers are adopting.
Getting to the root cause
Focused on studying host-pathogen interactions, Yang, an associate professor at Iowa State University in the department of Genetics, Development and Cell Biology, is continually searching for techniques and strategies to circumvent microbial infection of crops. Over the past several years, he has been using TALENs and CRISPR/Cas9 to short-circuit infection of rice with the microbial pathogen Xanthomonas oryzae.
Yang’s team creates expression vectors for the genome-editing reagents, transfects cultured rice cells using either a biolistic “gene gun” or genetically modified Agrobacterium, selects for transformants and then tests them for the presence or absence of the desired transgene. In all, he says, the process takes about four months from DNA delivery to testing. “That’s a limitation of working with plants,” he says.
On the surface, the process is otherwise little different from working with bacteria, yeast or mice. “DNA is DNA,” says Patrick Schnable, the Iowa Corn Endowed Chair in Genetics at Iowa State University. But there are some significant differences.
Researchers have two basic strategies for screening transgenic plants: PCR and DNA sequencing.
Yang uses both in his research, adopting PCR with transgene specific primers to identify positive clones and Sanger DNA sequencing of the resulting amplicons to verify their identity. “For the plant field, not many people use high-throughput next-generation DNA sequencing (NGS),” he says, largely because of cost considerations.
That’s not to say there isn’t a role for NGS, says Schnable. Among other things, it can be used after initial transgenic screening steps to ensure the genome is otherwise untouched by the genetic alterations that were made.
A third approach, useful when screening organisms modified by genome-editing strategies, is the T7 endonuclease assay. Here, genomic DNA surrounding the edited site is amplified and then denatured and renatured to form heteroduplexes between edited and unedited sequences. Addition of T7 endonuclease, which cuts at DNA mismatches, reveals the fraction of modified DNA in the reaction.
Harvest season
Whatever method researchers use, they first need to harvest genetic material. But unlike mammalian cells, plant cells can be tough to crack, literally. “When you get to cells from plants you have to deal with tougher structures,” says Michelle Mandrekar, a research scientist at Promega. There’s a cell wall, of course. And some plant tissues, such as stems and seeds, are quite sturdy.
As an added complication, plants are loaded with compounds, including terpenes, polyphenolics and polysaccharides, that can inhibit the enzymatic activities required by many molecular methods, such as those using DNA polymerases.
As a result, researchers typically use dedicated nucleic acid extraction and cleanup procedures when working with plants. One common approach involves freezing the plant material with liquid nitrogen, grinding it up with a mortar and pestle or by “bead beating” and then extracting the released cellular contents into a buffer, such as CTAB. Some researchers also use compounds such as polyvinylpolypyrrolidone (PVPP), to remove inhibitors.
A number of companies sell kits to simplify and automate plant nucleic acid extraction, for instance on columns or magnetic beads. These include Promega (the Wizard® Magnetic 96 DNA Plant System ), Takara (NucleoMag® 96 Plant), Thermo Fisher Scientific (ChargeSwitch® gDNA Plant Kit ) and QIAGEN (DNeasy Plant Mini Kit ).
Also available are reagents designed specifically to work with plant samples, such as Takara’s Terra DNA polymerase. “It has been developed to work with inhibitors, even without extraction of the DNA” from the plant sample, says François-Xavier Sicot, senior product manager at Takara Bio Europe.
Is it GMO?
When it comes to transgenic plants, there is the issue of regulatory screening to consider.
Many countries screen incoming foodstuffs to ensure no unauthorized genetically modified organisms (GMOs) enter the food supply, says Yalei Wu, senior staff scientist for genetic analysis research and development at Thermo Fisher Scientific.
Such screening regimes often address a two-tiered question, Wu says: Does a sample contain any genetically modified organisms, and if so, which ones? To answer the former question, Thermo Fisher Scientific’s TaqMan™ GMO Screening Kit uses real-time quantitative PCR to detect the presence of three promoters commonly used in plant transgenesis—the cauliflower mosaic virus 35S promoter, the figwort mosaic virus 34S promoter and the NOS terminator from Agrobacterium. To control for the possibility of an actual infection by one of these pathogens (as opposed to a transgenic material), the kit also includes primers for other sequences outside of these regulatory regions.
Given a positive result, regulatory officials can then look more deeply, comparing the sample’s genetic signature to a set of known transgenic designs using TaqMan assays designed specifically to detect them. Alternatively, scientists can use digital PCR, Wu notes—a strategy that offers the benefit of absolute quantitation without a standard curve.
Should they come up against a design they’ve never seen before, regulators can turn to sequencing, says Chris Moreland, global product manager for integrated solutions at Promega. “They can actually screen for non-authorized events that may not be of known sequence,” he says. One regulatory agency in Europe, he notes, is using NGS and targeted sequencing strategies to develop a screening library of known and newly discovered transgenic sequences. “It’s almost like a forensic application in that regard,” he says.
NGS offers an additional benefit, adds Wu: It enables researchers and regulators to trace the provenance of newly discovered GMO species, for instance to identify and trace cross-pollination or horizontal gene transfer events. “Our Ion Torrent platform is very good for studying parentage analysis with its targeted sequencing technology,” he says, “for figuring out where the plant came from.”
Given that many plants are polyploid, containing four or sometimes six copies of each chromosome, some plant researchers exploit long-read sequencing technologies—such as those from Pacific Biosciences and Oxford Nanopore—to distinguish the different copies of each chromosome, says Schnable. “The longer the reads are, the more likely we are to pick up a difference between genome A and genome B,” he notes.
Utilizing the latest molecular techniques, botanists continue to plant the seeds that are resulting in improved strains of crops. Regulatory agencies are using similar molecular approaches to ensure a better genetic understanding of these transgenic plants—and monitoring any safety issues and concerns associated with these new strains of “superplants.”
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