Table 1.

Comparative analysis of mutagenesis approaches for large-scale mutant analysis.

Mutagenesis Tools	Mutation Type	Advantages	Challenges	Precision	Scalability	References
Chemical (e.g., EMS)	Random	Simple non-transgenic with high mutagenesis efficiency	Requires extensive screening	Low	Low	[73]
T-DNA Insertion	Random	Genome-wide coverage; barcode-compatible. Thousands of mutants analyzed in parallel	Random insertion disrupts non-target genes.Individual mutant screening via PCR or phenotyping	Low	High	[24,28]
Transposons (e.g., Ac/Ds)	Random	Mobility enables regional mutagenesis.Reusable systems	Requires transposase control; lower efficiency Individual mutant screening via PCR and phenotyping	Moderate	Moderate	[74]
CRISPR/Cas9	Targeted knockouts/editing	High precision; multiplex editing of redundant genes	Complex design for large libraries	High	High	[64,75,76]
RNAi	Targeted knockdown	Rapid; scalable with barcoded vectors (e.g., pooled RNAi screens) for redundant genes	Requires efficient design for target silencing	High	Moderate	[41,64,77]
Activation tagging	Gain-of-function	Identifies dominant alleles	random activation may cause pleiotropy Low mutant frequency	Low	Moderate	[35,78]
FOX hunting	cDNA overexpression	Precise overexpression. Possible to use barcoding to trace back the cDNA clones; links phenotype to known genes	High cost of cDNA library construction. Complex phenotyping	High	High	[79,80]