The correct answer is (3) Mutagenesis with ethylmethane sulphonate.

What TILLING is

• TILLING = “Targeting Induced Local Lesions IN Genomes”.

• It is a reverse genetics technique that:

  • First creates random point mutations across the genome, typically by chemical mutagenesis.

  • Then uses PCR-based screening to identify mutations in a specific target gene.

• The most widely used chemical mutagen for TILLING is ethylmethane sulphonate (EMS), because it efficiently induces G:C → A:T point mutations that are easy to detect and can produce allelic series.​

Therefore, option (3) Mutagenesis with ethylmethane sulphonate is correct.

Why the other options are not used for TILLING

1. T-DNA tagging by Agrobacterium-mediated transformation

• T-DNA tagging is an insertional mutagenesis method, useful mainly for forward genetics or insertion knockouts.

• TILLING specifically refers to chemical mutagenesis + targeted screening of point mutations, not insertional tagging.

2. Transposon tagging using Ac/Ds elements

• Like T-DNA, Ac/Ds transposons create insertions, often used to disrupt genes or create reporter fusions.

• This is a different reverse/forward genetics tool and is not what TILLING denotes.

4. Protoplast transformation by electroporation

• Electroporation of protoplasts is a DNA delivery method, not a mutagenesis strategy.

• It does not, by itself, create random point mutations across the genome for TILLING.

So, among the given choices, only EMS mutagenesis (option 3) matches the definition and standard practice of TILLING.