Molecular Plant Breeding 2024, Vol.15, No.4, 167-177 http://genbreedpublisher.com/index.php/mpb 172 The development of Huaduo1 to Huaduo8 (H1-H8) involved crossing generations of autotetraploid rice, resulting in high pollen fertility (>92.29%) and stable chromosome configurations during pollen development (Guo et al., 2017; Bei et al., 2019; Chen et al., 2019; Ghaleb et al., 2020). Additionally, the identification of double neutral genes, such as San and Sbn, has been instrumental in overcoming pollen sterility barriers and achieving higher pollen fertility in autotetraploid rice hybrids (Chen et al., 2020). 4.3.2 Biotechnological approaches Genetic engineering, such as CRISPR technologies offer innovative solutions to address fertility issues in autotetraploid rice by targeting specific genes involved in meiosis and pollen development. The use of CRISPR/Cas9 technology to knock out specific genes, such as TMS9-1 andTMS5, has been shown to significantly impact fertility and pollen development. Mutants of these genes displayed low fertility and abnormal pollen development, indicating their crucial role in fertility regulation (Wu et al., 2020). Additionally, transcriptome analysis has identified DEGs associated with fertility, providing targets for genetic engineering to enhance fertility in autotetraploid rice (Guo et al., 2017). For example, the up-regulation of meiosis-related genes, such as RAD51D, and tapetal-related genes, such as MIL2, OsAP25, and OsAP37, has been associated with high fertility in newly developed tetraploid rice lines (Wu et al., 2020). Furthermore, the identification of meiosis-specific and meiosis-related genes through transcriptome analysis provides valuable insights into the molecular mechanisms underlying fertility restoration in autotetraploid rice (Wu et al., 2015; Guo et al., 2017). Moreover, transcriptome analysis has identified DEGs and specific molecular markers associated with fertility, such as meiosis-related genes and genes involved in photosynthesis and amino acid metabolis (Guo et al., 2017; Chen et al., 2020). These molecular markers can be used to screen and select fertile lines, thereby improving the efficiency of breeding programs. By integrating these breeding strategies, biotechnological approaches, and a deeper understanding of meiotic behavior, researchers can effectively overcome sterility barriers and enhance heterosis in autotetraploid rice hybrids. 5 Case Studies and Field Trials 5.1 Experimental design and methodology In the pursuit of overcoming sterility barriers and enhancing heterosis in autotetraploid rice hybrids, several experimental designs and methodologies have been employed. One notable study focused on the cytogenetics and transcriptome comparison between diploid and autotetraploid rice hybrids harboring double neutral genes, San and Sbn. This study involved detailed cytological and transcriptome analyses during meiosis and single microspore stages to understand the molecular mechanisms underlying pollen fertility in autotetraploid rice hybrids (Chen et al., 2020). Another significant approach utilized CRISPR/Cas9 technology to edit the TMS5 gene in neo-tetraploid rice, developing thermo-sensitive genic male sterile (TGMS) lines. These lines were then crossed with other tetraploid rice lines to generate F1 hybrids, which were evaluated under both controlled and natural growing conditions (Chen et al., 2022; Samonte et al., 2023). Additionally, previous studies have demonstrated that the sterility of F1 hybrids in autotetraploid rice is primarily due to embryo sac abortion, which is also influenced by the genotype of the S5 gene (Fimanekeni et al., 2023). 5.2 Examples of successful autotetraploid hybrids Field trials have demonstrated promising results in overcoming sterility barriers and enhancing heterosis in autotetraploid rice hybrids. Several studies have demonstrated significant yield improvements in autotetraploid rice hybrids. For example, hybrids such as T461A/T4002 and T461A/T4193 have shown yield increases of 46.3% and 38.3%, respectively, compared to the commercial diploid hybrid Shanyou 63 (Tu et al., 2007).
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