Molecular Plant Breeding 2024, Vol.15, No.6, 429-441 http://genbreedpublisher.com/index.php/mpb 437 6.4 Field trials and phenotypic analyses Field trials and phenotypic analyses are essential for validating the effects of genetic modifications and understanding the practical implications of gene expression changes under natural environmental conditions. For example, CRISPR/Cas9-mediated gene mutagenesis in maize and rice has been followed by phenotypic and cytological analyses to identify new GMS genes and understand their roles in anther and pollen development (Jiang et al., 2021; Chen et al., 2020; Yan et al., 2024). These field trials help in assessing the stability and effectiveness of the genetic modifications in real-world agricultural settings. By integrating these experimental approaches and methodologies, researchers can gain a comprehensive understanding of how environmental factors regulate the expression of male sterility genes in rice, ultimately aiding in the development of improved crop varieties. 7 Future Directions and Perspectives 7.1 Potential for developing climate-resilient MS lines The development of climate-resilient MS lines in rice is crucial for ensuring stable two-line hybrid seed production under varying environmental conditions. Research has shown that the regulation of MS in rice is highly sensitive to temperature, photoperiod, and humidity changes. For instance, the identification of miRNAs such as miR156, miR5488, and miR399, which influence male fertility by affecting SPLs, lignin synthesis, and flavonoid metabolism, provides a new understanding of the regulatory mechanisms involved (Sun et al., 2021b). Additionally, the discovery of TGMS loci and their associated regulatory mechanisms offers potential targets for breeding thermo-tolerant rice species without yield penalties (Kan and Lin, 2021; Lee et al., 2005; Liu et al., 2010; Zhang et al., 2022; Lin et al., 2023). Future research should focus on identifying and characterizing more such loci and understanding their interactions with environmental factors to develop robust MS lines. 7.2 Advances in precision breeding and genetic engineering Advances in precision breeding and genetic engineering hold significant promise for the development of MS lines in rice. Techniques such as CRISPR/Cas9 can be employed to edit specific genes involved in MS regulation, such as the p/tms12-1 locus, which has been shown to control P/TGMS through a point mutation in a noncoding RNA (Zhou et al., 2012; Ding et al., 2012). Moreover, transcriptome and metabolome analyses have revealed that genes involved in sugar, lipid, and phenylpropanoid metabolism are essential for male fertility, providing additional targets for genetic manipulation (Sun et al., 2022). By leveraging these advanced techniques, it is possible to create rice lines with precise and stable MS traits, enhancing the efficiency of hybrid seed production. 7.3 Importance of multi-disciplinary approaches The complexity of MS regulation in rice necessitates a multi-disciplinary approach that integrates genetics, molecular biology, environmental science, and breeding strategies. For example, the cooperative regulation of major-effect sterile genes with photoperiod-,,temperature-, and humidity-sensitive genes, as well as the role of minor-effect genes in determining the critical temperature of sterility transition, highlights the need for a comprehensive understanding of the genetic and environmental interactions (Chen et al., 2010; Ding et al., 2012; Yan et al., 2024; Chen et al., 2020). Additionally, the integration of transcriptomic, metabolomic, and electron microscopy analyses has provided valuable insights into the molecular and physiological processes underlying MS (Sun et al., 2022). Collaborative efforts across these disciplines will be essential for developing innovative solutions to the challenges posed by environmental variability. 7.4 Anticipated challenges and solutions Several challenges are anticipated in the development and application of MS lines in rice. One major challenge is the variability in environmental conditions, which can affect the expression of MS genes and compromise hybrid seed production. For instance, the response of TGMS lines to varying temperature situations has been shown to be influenced by the genetic background of the recipient lines and the duration of temperature exposure (Viraktamath and Virmani, 2001; Lin et al., 2023; Yan et al., 2024). To address this, hybrid rice breeders need to develop genetically diverse TGMS lines with critical sterility-inducing temperatures that are resilient to sudden temperature fluctuations. Another challenge is the potential yield penalties associated with breeding
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