Maize Genomics and Genetics 2025, Vol.16, No.2, 80-88 http://cropscipublisher.com/index.php/mgg 85 In addition, the combination of high-throughput and automated systems has also improved induction efficiency. For example, in the Stock6-derived induction line, the improved CENH3 gene was overexpressed, and the maternal haploid induction rate was increased to 16.3% (Meng et al., 2021). Combining gene editing and advanced instruments not only speeds up the breeding process, but also improves the accuracy and stability of induction, laying a solid foundation for large-scale and efficient corn breeding. 6 Challenges and Limitations 6.1 Low Induction Rate In haploid breeding, a very troublesome problem is that the haploid induction rate (HIR) is generally low. To say how important this is, the efficiency of haploid induction directly affects whether the double haploid (DH) technology can be carried out smoothly. But the reality is that it is still difficult to achieve a high HIR. In fact, the genetic factors that control haploid induction are quite complex, involving several quantitative trait loci (QTL), such as qhir1 and qhir8, both of which are closely related to HIR. For example, the MTL (also called ZmPLA1 or NLD) gene mutation in the qhir1 region can bring about an induction rate of about 2%, but this value is far from enough for modern breeding, because modern breeding generally hopes that the HIR can reach about 10% (Prigge et al., 2012; Zhong et al., 2019). Of course, some people have tried to improve this indicator through genetic modification, such as overexpression of modified CENH3, which does seem promising, but after all, these methods have not been widely used (Meng et al., 2022). In addition, don't forget that the genetic background of the environment and variety will also affect the results. The efficiency of haploid induction varies greatly among different maize lines, which makes it more complicated to maintain high HIR (Dwivedi et al., 2015; Liu et al., 2019). Because of these unstable factors, researchers have been working hard to find genetic factors that are more suitable for different germplasms to make haploid induction more robust. 6.2 Genome stability Another problem is how to ensure that the genome is not damaged during haploid induction. After haploid formation, chromosomes must be doubled, but this process sometimes brings genetic instability, affecting the quality of the final DH line. Although colchicine is a commonly used chromosome doubling drug with good results, it is toxic after all and may cause some unexpected genetic changes. What is more troublesome is that manipulating key genes such as MATRILINEAL (MTL) sometimes causes off-target effects and indirectly destroys genome stability (Kelliher et al., 2017). In addition, some new haploid identification markers developed in recent years, such as red root markers and high oil markers, can improve efficiency, but they should not be taken lightly. It is necessary to ensure that these markers do not bring additional genetic variation, otherwise the gains will outweigh the losses (Chaikam et al., 2019). After all, ensuring the genome integrity of the DH line is the key, and any genetic instability will weaken the effect of DH technology in breeding superior lines. 6.3 Economic and technical feasibility When it comes to promoting haploid breeding technology, economic and technical barriers cannot be ignored. The establishment and maintenance of haploid induction lines themselves are not small, and the haploid identification and chromosome doubling require specialized equipment and technical support. This is a great cost pressure for many breeding programs, especially those in developing countries (Dwivedi et al., 2015; Chaikam et al., 2019). This high threshold makes it difficult for small-scale breeding teams to do this. The technical complexity is also high. Although the automation technology of haploid identification is improving, which can theoretically reduce time and cost, these technologies are still in the early stages and cannot be used in many projects (Chaikam et al., 2019). At the same time, combining gene editing tools (such as CRISPR/Cas9) with haploid induction can indeed accelerate the breeding process, but the related technical costs and professional requirements are also high, and not all breeding teams can easily handle them (Wang et al., 2019).
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