4. Application value evaluation of GMS genes and mutants in crop plants

Compared to CMS and environment-sensitive genic male sterility (EGMS), GMS has many advantages such as the high germplasm utilization efficiency, higher male-sterility stability under various environments, and lower linkage rate with adverse traits. As more and more GMS genes have been cloned in crops, the BMS systems by using GMS gene have been developed in several crop plants and come into commercial utilization, such as SPT and MCS systems [3]. However, before utilization in the BMS systems, many characteristics of GMS gene and its mutant should be assessed systemically, such as genetic stability analysis of male-sterility, heterosis comparison, and analysis of potential linkage with bad traits.

### 4.1 Genetic stability analysis of male-sterility

Firstly, the genetic stability of the male-sterile mutant should be appraised in different genetic backgrounds and various environments. The general procedure is as follows (Figure 5A; the recessive ms mutant is taken as an example): a homozygous ms mutant is used as female parent and crossed with hundreds of inbred lines with broad genetic backgrounds to get the heterozygous F1 hybrids. Then one of the F1 hybrids is self-pollinated to produce F2 seeds. Thereafter 50–100 of the F2 seeds from each cross are grown in various environments. The fertility segregation ratios

#### Figure 5.

The application value evaluation of GMS genes and mutants in crop plants.

Molecular Cloning of Genic Male-Sterility Genes and Their Applications for Plant Heterosis… DOI: http://dx.doi.org/10.5772/intechopen.86976

of the F2 populations are investigated, and anthers of three sterile individuals in each F2 population are collected and stained with 1% I2-KI solution to examine male-sterility status of pollen grains. If the segregation ratio of fertility to sterility in all crosses shows 3:1 as expected, we can say that the male sterility is genetically stable in different genetic backgrounds and various environments. Otherwise, if the ratio is not always 3:1 and confirmed by the molecular marker-assisted selection results, we can say that the male sterility is unstable in different genetic backgrounds and/or various environments. For instance, the male-sterility stability of maize ms30-6028 mutant was analyzed by crossing with 329 maize inbred lines and observation of the segregation ratio of fertility to sterility in F2 populations, suggesting that ms30-6028 is a stable male-sterility mutant under diverse genetic backgrounds [18].

#### 4.2 Heterosis comparison between ms mutant and wild type

Secondly, the effects of ms mutant on heterosis should be analyzed by comparing the yield and related agronomy traits between F1 hybrid plants produced by using ms mutant and wild type (WT) as female parents and crossing with the same inbred line (Figure 5B). For instance, to test whether ms30-6028 gene affects maize heterosis and grain yield, ms30-6028 mutant and its homozygous WT line were used as female parents and crossed with 30 maize inbred lines, respectively. The harvested F1 hybrids and their corresponding parental lines were grown according to the planting model of maize field production in two different locations. Eighteen agronomic traits such as plot yield, whole growth period, plant height, ear height, and hundred-kernel weight were investigated to compare the differences of heterosis and field production performance of 30 pairs of hybrid combinations using ZmMs30 and ms30-6028 homozygous plants as female parents, respectively. The results indicated that ms30-6028 mutation has no obvious negative effects on maize heterosis and field production, suggesting that ZmMs30 gene and its mutant ms30-6028 are applicable for hybrid maize breeding and hybrid seed production [18].

#### 4.3 Analysis of potential linkage with disadvantage genes and traits

Furthermore, other than the male-sterility stability analysis and heterosis comparison described above, the potential linkage with bad traits of ms locus should be analyzed. There are at least two ways to get this target: one is phenotypic observation of the hybrid plants that come from the homozygous ms mutant used as female parent, while those of the fertile sibling used as control. If the field production performances of the hybrid plants between ms mutant and WT are similar with each other, we can say that the ms mutation is not linked with disadvantage traits and thus can be applicable in hybrid seed breeding and production. For instance, maize Ms44 hybrids showed no yield penalty in any of the tested environments, indicating that it is desirable for commercially viable products [22]. The other is sequencing of the putative genes near the ms locus and screening for the potential disadvantage genes based on bioinformatic analysis.
