Among the most effective ways to assign functions to genes is by having genetic variants of the gene in question. Such genetic variants can be of natural origin or generated deliberately by mutagenesis. The effectiveness and use of induced mutagenesis depends a great deal on the mutation efficiency of the reagent and/or the procedure. Various forms or reagents to intentionally induce mutations in plants exist, but chemical mutagenesis by ethyl methanesulfonate (EMS), an inducer of point mutations in DNA, has gained interest. EMS is an alkylating agent that reacts mainly with the guanine (G) base in the DNA to change it to O-6-ethylguanine, which prefers to base pair with adenine (A) during DNA replication instead of its natural partner, the cytosine (C). As a result, an A is introduced at the location in the DNA where there was a C before. Because the new A will naturally hydrogen bond with thymine (T) during further rounds of DNA replication, the original GC base pair is converted into an AT base pair transition when mutagenesis is done with EMS.
Seed mutagenesis is the method of choice in almost all plant species except maize, where EMS mutagenesis is done via pollen. The main reason for this is that maize is a monoecious plant with separate male and female inflorescences at two separate locations on the plant. The male flowers are present only in the tassel, which forms at the top of the main axis of the plant, and the female flowers are present in the ear, which is a modified lateral shoot on the side of the main axis approximately 5-6 leaves below the tassel node. The maize embryonic cells that eventually morph into the tassel or the ear are already split into separate lineages by the time the maize kernels (seed) mature. When the maize seed is treated with EMS, the mutations that are generated in the ear primordial cells are completely independent from the mutations that are induced in the tassel primordial cells. To get these mutations in homozygous condition, which is absolutely needed to reveal recessive mutations, the M3 generation in maize is of interest because even when M1 plants (derived from EMS treated seed) are self-fertilized to produce the M2 progeny, the ear and tassel mutations are consolidated together in a heterozygous condition. This reproductive peculiarity of maize also causes the original germ line mutations to be amplified enormously, especially via the pollen, imposing serious limitations on the recovery and genetic interpretation of most mutations.
To eliminate these problems with seed mutagenesis, as well as to avoid the tremendous amount of work and expense that goes with having to raise an extra generation to recover mutant phenotypes (propagating thousands of plants, especially by self-pollination, is not trivial), a pollen mutagenesis protocol involving EMS was developed. In addition to allowing the revelation of dominant mutants in the M1 generation and recessive mutations in the M2 generation, this pollen mutagenesis protocol generates mutations that are independent of each other. An additional advantage of mutagenesis via pollen is that it allows one to conduct targeted mutant screens (also known as non-complementation screens) to specifically generate a series of mutations in a gene of interest, for example screening for non-complementation of a recessive mutation.
One disadvantage of pollen mutagenesis, which has been optimized to work only in maize thus far, is its mutation efficiency. It is about one out of 1,000 gametes treated with EMS, when gauged by functional genetic tests. When tested molecularly, the density of mutations caused by the pollen EMS protocol in maize was found to be about 2 mutations per Mb of DNA. Whatever the reasons, the relatively low mutation frequency of mutagenesis in this protocol acts as a deterrent for many working either in the public or commercial sector. This impediment can be eliminated if the mutation frequency is enhanced.
There is therefore an unmet need for a cost-effective, high accuracy method for generating independent mutant alleles.