How a 25-Year-Old Technology Defies the Gene Editing Revolution
Imagine wanting to understand what a single gene does in a crop plant. Do you turn to the precision of CRISPR gene-editing scissors? Or do you reach for a classic, random method that has been fueling discovery for decades? This is the central question in plant science labs today.
At its heart, TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful "reverse genetics" strategy 1 . First developed in 2000, it begins with creating random mutations across a plant's entire genome, typically using chemical mutagens 4 . Scientists then sift through this mutated population, like searching for a needle in a haystack, to find individual plants with specific, desirable changes in a gene they are interested in 1 .
These resulting mutant plants are non-transgenic, meaning they are not considered genetically modified organisms (GMOs) in most jurisdictions, which allows them to be directly used in pre-breeding programs 1 4 .
| Term | Field | Primary Function | Key Feature |
|---|---|---|---|
| TILLING (Targeting Induced Local Lesions IN Genomes) | Plant Genetics & Genomics | Identifies specific mutations in genes of interest within a randomly mutated population 1 | A reverse genetics method for gene function discovery and crop improvement 7 |
| Soil Tillage | Agronomy & Soil Management | Prepares soil for planting by mechanical agitation (e.g., turning, stirring) | An agricultural practice for seedbed preparation and weed control 5 |
| CRISPR/Cas9 | Genetic Engineering & Biotechnology | Enables precise, targeted edits to the DNA sequence at a specific location in the genome 1 | A site-specific genome editing tool |
TILLING mutants are not subject to GMO regulations, allowing faster application in breeding programs.
Starts with a gene sequence and works to find associated phenotypes, unlike forward genetics.
TILLING's power lies in its two-step process: first, creating a vast library of random genetic variants, and second, using sophisticated technology to find the one-in-a-million variant that matters.
The process begins with mutagenesis. The most common method uses a chemical called EMS (Ethyl Methanesulphonate) 1 . When seeds are soaked in EMS, it causes random point mutations throughout the plant's DNA as the seeds germinate and grow.
These first-generation plants (M1) are grown and self-pollinated. The seeds they produce (M2 generation) are the true treasure trove, as these contain stable mutations that can be passed on. A single TILLING population can comprise thousands of these M2 plant lines, each a unique bundle of genetic variations 1 .
Once a population is established, the hunt begins. If a scientist wants to study a specific gene, they design molecular "probes" to match its sequence. High-throughput methods are then used to screen the entire population's DNA.
Early TILLING platforms used enzymes like CEL I to scan for and cut DNA at locations where mutations created a mismatch 1 . Today, "TILLING by Sequencing" represents a major technological leap 7 . With this method, the target genes from all the mutant plants are sequenced simultaneously using next-generation sequencing technologies.
Seeds are treated with EMS to induce random mutations throughout the genome.
Treated seeds grow into first-generation plants that are self-pollinated.
Seeds from M1 plants create a population with stable, heritable mutations.
DNA is extracted from M2 plants and screened for mutations in target genes.
Promising mutants are grown and phenotyped to confirm trait changes.
To see TILLING in action, look no further than a 2025 study aimed at improving the taste and nutritional quality of soybeans 7 .
Soybean seed quality is heavily influenced by its sugar content. Sucrose is desirable because it provides sweetness and is a key energy source for fermenting soy products like tofu and natto.
In contrast, other sugars like raffinose and stachyose are considered "antinutritional" because humans and monogastric animals cannot digest them, leading to gastrointestinal issues 7 .
The goal was clear: find soybean mutants with higher sucrose and optimal levels of raffinose and stachyose.
| Mutant Line | Gene Affected | Sucrose Content |
|---|---|---|
| SL446 | Glyma.02G240400 | 9.5% |
| F1115 | Glyma.02G240400 | 9.1% |
| Wild-Type | - | 5-7% |
Behind every successful TILLING project is a suite of essential research reagents. The following details the key components that make this technology possible.
| Reagent / Material | Function in TILLING Process | Explanation |
|---|---|---|
| EMS (Ethyl Methanesulphonate) | Chemical Mutagen | An alkylating agent that causes random point mutations throughout the plant genome, creating genetic diversity 1 |
| CEL I / Surveyor Nuclease | Mutation Detection | Enzymes used in early TILLING to detect and cleave DNA at the site of base pair mismatches 1 |
| PCR Reagents | DNA Amplification | Used to amplify the specific gene region of interest from mutant plant DNA 7 |
| NGS Kits | Mutation Discovery | The core of "TILLING by Sequencing"; allows high-throughput sequencing of target genes 7 |
| Bioinformatics Software | Data Analysis | Crucial for analyzing sequence data and accurately pinpointing mutations |
EMS creates random point mutations (mainly G/C to A/T transitions) across the genome.
Advanced screening methods identify specific DNA changes in target genes.
Bioinformatics tools process sequencing data to pinpoint mutations accurately.
So, does TILLING still have a place in the age of CRISPR? The scientific literature suggests a resounding yes. Instead of being rendered obsolete, TILLING is finding its niche in a complementary relationship with newer technologies.
The era of TILLING is far from over. While the dazzling rise of CRISPR has rightfully captured the scientific imagination, TILLING has cemented its role as a robust, accessible, and regulatory-friendly method for generating genetic diversity.
The question is not whether TILLING or CRISPR is the "winner," but how these technologies can work in concert. A researcher might use TILLING to discover a novel and valuable genetic variant in a wild crop relative and then use CRISPR to precisely introduce that same variant into an elite breeding line.
As recent reviews conclude, based on recent achievements, "TILLING still plays an important role in plant research as a valuable tool for generating genetic variation for genomics and breeding projects" 1 4 .
TILLING, a seasoned veteran, is proving it still has plenty of turns left.