Table 3.

Reverse genetics methods used for functional genomics in plants.

Method	Advantages	Disadvantages	References
RNA interference	▪ High-throughput vectors ▪ Heritable, ▪ Ability to silence multiple target genes at once ▪ Single copy of the target transgene is frequently sufficient to induce silencing	▪ Variability in silencing efficiency ▪ Require efficient transformation ▪ Not developed for all species ▪ Effect on non-target genes	[100-102]
Virus-induced gene silencing (VIGS)	▪ No limitation imposed by transformation efficiency ▪ Rapid, easy to use ▪ Can be adapted for high-throughput screens ▪ Generate partial loss-of-function	▪ Host range limitations ▪ Not established on all plant species ▪ Allows only transient expression ▪ Silencing level is variable	[8, 100, 103, 104]
Ectopic expression	▪ Can be adapted for high-throughput screens ▪ Suitable for transgene expression and gain-of-function analysis	▪ Limited to transformable plant species ▪ Possibility of generating misleading neomorphs ▪ Expression level can be changed by exogenous regulatory sequences	[1, 4, 11, 12]
Target Induced Local Lesions in Genome (TILLING)	▪ Stable mutations ▪ Suitable for non-transformable species ▪ Allows identification of allelic series of mutants with a range of modified functions for a particular gene	▪ Low/medium-throughput ▪ Desired mutation might never be found ▪ Need a large mutant population ▪ Relatively expensive	[1-3, 105, 106]
Insertional mutagenesis	▪ High-throughput ▪ Results in stable mutations ▪ Suitable for non-transformable species ▪ Can be adapted for both loss-of-function and gain-of-function studies	▪ Desired mutation might never be found ▪ Variable effects depending on site of integration	[1, 2, 107, 108]
Gene targeting and Genome editing	▪ Highly specific ▪ Most revolutionary method that can be used for gene replacement ▪ Can be adapted for both loss-of-function and gain-of-function studies	▪ Very low efficiency of homologous recombination	[1, 5, 9, 109]