1 Introduction

It goes without saying that corn (Zea mays L.) is an indispensable member in the fields of food, feed and fuel. Millions of people around the world rely on it as their staple food. It is one of the most important cereals for mankind, second only to rice and wheat (Gupta et al., 2022; Wang et al., 2022). But in many places, especially in tropical agricultural areas where people rely on the weather for food, it is not so easy to grow corn. Drought, heat, high humidity, and saline-alkali land are all long-standing problems that affect harvests (Prasanna et al., 2020; 2021). Can we use existing methods to combat these climate troubles? It is difficult. Therefore, we have to think of new ways, and the upgrading of breeding technology has become urgent.

In this regard, haploid breeding is a method that has attracted much attention, especially the double haploid (DH) technology, which is considered a major breakthrough in the breeding process. Its principle is actually not complicated: first produce a haploid plant with only one set of chromosomes, and then use artificial means to double its chromosomes to become a homozygous DH line. Because there is no need for generation after generation of screening, this step directly increases the breeding speed, which is especially convenient when doing hybrid breeding (Dwivedi et al., 2015; Chaikam et al., 2019; Meng et al., 2021). Of course, this technology alone is not enough. In recent years, people have begun to use it in combination with molecular markers, genome editing and other technologies, which not only improves efficiency, but also broadens its application range in commercial breeding (Wang et al., 2019; Gupta et al., 2022; Zhou and Jiang, 2024).

This review is intended to review the current progress in haploid maize breeding. The focus is not only on DH technology itself, such as how to improve the success rate of haploid induction, how to quickly identify haploids, and how to embed gene editing tools such as CRISPR/Cas9, etc. More importantly, we want to see whether these technologies can be used in actual stress-resistant breeding, especially under extreme conditions such as high temperature and drought. As for whether they can increase genetic gain and improve breeding efficiency, it depends on their performance in reality.

2 Overview of Haploid Breeding Technology

2.1 Haploid induction type

Not all corns will spontaneously produce haploids under natural conditions. This situation is actually very rare, and the incidence rate is usually less than 0.1% (Liu et al., 2017; Zhou Liang, 2024). However, this type of "natural induction" does exist, although it is almost impossible to rely on it to promote breeding.

In contrast, artificial means are obviously more reliable. The most commonly used one is a haploid induction system. For example, the type derived from Stock6 can increase the haploid production rate to 1%~2% (Liu et al., 2017). Its operation is actually not complicated: pollinate the target plant with pollen from the induction system, and haploids may appear in the offspring. If some molecular tools are used, such as manipulating the key protein CENH3, through overexpression and other methods, the induction efficiency can be further improved (Meng et al., 2022). It can be said that haploid induction has changed from "relying on nature" to "relying on technology".

2.2 Historical Development

Corn haploid breeding is not a new invention in recent years. As early as the late 1950s, scientists discovered a key material-Stock6 inducer line (Kelliher et al., 2017). It was this material that opened the door to artificial induction of haploids.

Since then, the in vivo induction system has gradually formed and been used, becoming a standard tool for many breeding programs. In the following decades, technological progress has continued, especially the introduction of double haploid (DH) technology, which allows people to obtain homozygous lines faster and greatly speeds up the pace of breeding (Chaikam et al., 2019; Meng et al., 2021). Further breakthroughs have appeared in the understanding of the mechanism. For example, the discovery of the MATRILINEAL (MTL) gene not only improves the induction efficiency, but also makes us more clear about how this process occurs (Kelliher et al., 2017).

2.3 Current mainstream methods

Today, when breeding haploids, everyone basically chooses those optimized inducer lines. Stock6 materials are still the main force. They are not only highly efficient in induction, but also highly adaptable and suitable for use in different environments and breeding goals (Chaikam et al., 2019; Trentin et al., 2020). Of course, breeding now relies on more than "traditional methods". Molecular technology is also used in many experiments. For example, methods such as genome editing and marker-assisted selection have been integrated into the haploid breeding process. There is also a system called IMGE, which combines CRISPR/Cas9 technology with haploid induction, which is more efficient and targeted (Wang et al., 2019). This set of solutions is now gradually recognized, especially when you want to quickly obtain edited materials for a certain trait (Andorf et al., 2019; Jacquier et al., 2020).

3 Production of Double Haploid Maize

3.1 Concept and advantages

The term "pure line" is not unfamiliar in breeding, but after using double haploid (DH) technology, the speed of obtaining homozygous materials has been completely refreshed. Traditional methods often rely on self-pollination from generation to generation to slowly advance, while the DH method directly doubles the number of chromosomes in haploid cells to obtain completely homozygous individuals. The theory sounds complicated, but the purpose is actually very direct: to obtain genetically consistent materials as soon as possible (Figure 1) (Chaikam et al., 2019; Meng et al., 2021).

Figure 1  Model of the rapid breeding of DH lines by the use of inducer lines containing purple markers or fluorescent markers (Adopted from Meng et al., 2021) Image caption: The embryos that lack purple color or fluorescent signals are haploid. They are selected to put into N6 culture media for carrying out the subsequent doubling process with the chemical reagents. N6: short for N6 culture media (Adopted from Meng et al., 2021)

However, it is not just "fast". In the breeding process, DH lines have several benefits that cannot be ignored. For example, they are genetically stable and do not require repeated screening; for example, planting management is also easier because they are uniform. For hybrid seed production, this consistency is particularly important. After all, if the parents are unstable, it is difficult to say how the offspring will perform (Chaikam et al., 2019; Jacquier et al., 2020). Of course, DH is not a panacea, but once used in combination with molecular markers, the genetic gain it brings is another bonus (Chaikam and Prasanna, 2020; Meng et al., 2021).

3.2 Key steps in production

In the final analysis, making a DH line is actually a process of turning a "haploid" into a "homozygous", and the operation steps are relatively fixed. First, haploids must be induced, which is usually done with the help of a specific male genotype; then, haploids must be distinguished from normal diploids in seeds or seedlings, and this step cannot be wrong. Commonly used methods include identification systems such as red root markers and high oil markers. Although there are many means, accuracy is always the first priority (Chaikam et al., 2019; Jacquier et al., 2020).

The identified haploids will undergo chromosome doubling treatment, commonly using chemical agents such as colchicine. This step is critical because only after the number of chromosomes is doubled can the plant be restored to fertility. After that, we can get the homozygous lines we want through self-pollination (Chaikam et al., 2019; Chaikam and Prasanna, 2020).

Of course, the current technology is much better than before. For example, the new generation of induced lines not only has high induction efficiency, but also can adapt to tropical or local materials; and the emergence of automated identification systems has also made some repetitive tasks much easier. Improvements in chromosome doubling have also played a significant role in saving costs and time (Chaikam et al., 2019; Jacquier et al., 2020).

3.3 Current applications

Today, DH technology has become an indispensable part of the corn breeding process. Especially in the development of hybrid varieties, quickly obtaining homozygous lines is almost equivalent to locking in the possibility of excellent combinations in advance. Once the cycle is shortened, the breeding efficiency will naturally increase (Wang et al., 2019; Meng et al., 2021). Some breeding projects even use DH directly in recurrent selection, with good results (Gallais and Bordes, 2007).

In recent years, DH technology has been continuously "upgraded". For example, some teams have tried to combine it with genome editing technology to come up with the so-called IMGE (haploid induction-mediated genome editing) method-this is actually to use CRISPR/Cas9 directly on haploids, bypassing those tedious traditional steps, and quickly obtain homozygous for the target trait (Wang et al., 2019; Jacquier et al., 2020). Combined with tools such as molecular markers or genomic selection, the entire breeding process is more accurate and efficient (Chaikam and Prasanna, 2020; Meng et al., 2021).

4 Molecular and Genetic Progress in Haploid Induction

4.1 Discovery of key genes

The study of haploid induction has made important progress, and some key genes discovered have brought new possibilities for corn breeding. For example, the MTL (also called ZmPLA1 or NLD) gene, although its loss triggers haploid induction, this was not discovered by chance. Through a large number of genetic studies, this gene was confirmed to play an important role in the haploid induction process (Liu et al., 2019). On the other hand, the discovery of the ZmDMP gene used a different method - map-based cloning, and later it was knocked out by CRISPR-Cas9 technology, and it was found that single nucleotide mutations can increase the haploid induction rate by two to three times, which is very helpful for improving efficiency (Figure 2) (Zhong et al., 2019). Despite this, the genetic mechanism of haploid induction is still complex, and these genes are only the key part of it.

Figure 1 Phenotypic evidence of transgenic events that enhanced HIRs (Adopted from Zhong et al., 2019) Image caption: a, Bar plot of the HIR of ZD958 ears pollinated by genotype classes A, H and B derived from transgenic events T0-15 and T0-17. b, Bar plot of the EnA rate (EnAR) of ZD958 ears pollinated by genotype classes A, H and B derived from transgenic events T0-15 and T0-17. n indicates the number of ears used for calculating the HIR and the EnAR of each genotype. c, Performance of ZD958 ears pollinated by genotype classes A (n=30), H (n=46) and B (n=63). Scale bar, 2 cm. Genotype classes A, H and B represent the genotype combinations zmpla1-ZmDMP, zmpla1-heterozygous and zmpla1-(zmdmp-ko), respectively (a-c). d, The blue bar plot shows the fold change of the HIR and the EnAR between genotype classes zmpla1-zmdmp (natural allele) and zmpla1-ZmDMP in the CAU5-CAUHOI F2 population; the orange bar plot shows the fold change of the HIR and the EnAR between zmpla1-(zmdmp-ko) (knockout allele) and zmpla1-ZmDMP in the F2 population. n indicates the number of F2 population. Data represent the mean ± s.d.; **, P< 0.01, ***P< 0.001 (two-sided Wilcoxon rank-sum test) (a,b,d) (Adopted from Zhong et al., 2019)

4.2 Application of molecular markers

Molecular marker technology has become a key tool to improve the efficiency of haploid induction. It helps locate genomic regions associated with DH production, thereby speeding up the screening process (Dwivedi et al., 2015). For example, traditional red root markers and high oil markers are now integrated into haploid induction lines, which enables DH technology to be better applied to diverse maize germplasms, such as flint maize and tropical materials, because traditional markers such as R1-nj do not perform well in these materials (Chaikam et al., 2019).

In addition, some transcription factors such as ZmC1 and ZmR2 have been used to develop new haploid identification systems. They can activate anthocyanin synthesis in the embryo and aleurone layer, greatly improving the speed and accuracy of identification. The new haploid inducer line MAGIC1 combines these markers and can identify haploids nine days earlier than traditional methods with an accuracy of up to 99.1% (Chen et al., 2022). This progress reduces breeding time and cost, which is very important for large-scale production of DH lines (Chaikam et al., 2019).

4.3 Application of gene editing

Speaking of gene editing, CRISPR/Cas9 technology has completely changed the face of haploid breeding. Through haploid induction-mediated genome editing (IMGE), scientists can use inducer lines carrying CRISPR/Cas9 to directly modify the desired agronomic traits in corn, so that homozygous improved DH lines can be obtained within two generations, avoiding the lengthy steps of traditional breeding (Wang et al., 2019).

5 Innovation of Haploid Induction Scheme

5.1 Chemical induction method

In the past, chemical doubling agents were a common means to improve the efficiency of haploid induction in maize. Although effective, these chemical reagents pose considerable safety hazards to operators and plants themselves, and the common doubling rate is about 10% to 30%. In recent years, researchers have become increasingly interested in a method called spontaneous haploid genome doubling (SHGD), which does not rely on chemicals, but has an unstable success rate, ranging from less than 5% to more than 50%. The latest genetic mapping studies have discovered some key quantitative trait loci (QTLs), which are expected to improve the success rate of SHGD (Figure 3) (Boerman et al., 2020).

5.2 Environmental optimization

Environmental factors play a significant role in the success or failure of haploid induction. Conditions such as temperature, humidity, and light will significantly affect the haploid induction rate (HIR). Corn with different genetic backgrounds also responds very differently to the environment. For example, RWS and Mo-17 induction lines perform better under certain environmental conditions, while sweet corn and flint corn usually have lower induction rates (Trentin et al., 2022).

Studies have found that the interaction between the induction line and the donor background is particularly important. Some donor backgrounds contain anthocyanin repressor genes, which will increase the haploid misjudgment rate and reduce HIR (Trentin et al., 2022). Therefore, it is not enough to adjust the environment alone, and the optimization plan must be customized in combination with genetic factors. By reducing the negative impact of these genetic and environmental factors, breeders can design more stable and effective haploid induction methods to maximize efficiency under a variety of conditions.

5.3 Advanced Instruments

With technological advances, advanced instruments and automation technologies are revolutionizing the way haploid induction is performed. Automation technology, especially in haploid identification, has greatly simplified the process. New marker systems, such as red root markers and high oil markers, are increasingly used in induction lines, making it easier and more accurate to distinguish haploids from diploids (Chaikam et al., 2019). These new markers are particularly useful for germplasms where traditional markers such as R1-nj have failed, broadening the application range of DH technology.

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).

7 Case studies and Field Applications

7.1 Commercial case studies

Double haploid (DH) technology is now a key tool in commercial corn breeding, mainly because it can quickly and efficiently obtain homozygous lines. Compared with traditional inbred line breeding, this technology has obvious advantages in economic cost, operation process and genetic stability. However, the popularization of DH technology is not achieved overnight. For example, the development of induced lines with high haploid induction rate and the adaptation of these lines to different environments, especially tropical regions, have greatly promoted its application (Chaikam et al., 2019).

It is also worth noting that the introduction of new marker systems, such as red root markers and high oil content markers, has made DH technology more efficient in dealing with diverse germplasm resources. The germplasm covers flint corn, local varieties and even tropical materials, and the utilization rate of these diverse gene pools has been improved (Chaikam et al., 2019).

In addition, DH technology has also performed well in the development of hybrids. Through in situ parthenogenesis, DH lines can be generated in large quantities and quickly, and these inbred lines become the basis of hybrid parents. This method is not only low-cost but also highly efficient, which accelerates the pace of hybrid breeding. More importantly, the genetic progress obtained through DH-RS has been able to match the traditional methods, showing the important value of DH technology in commercial breeding (Gallais and Bordes, 2007).

7.2 Field trials and performance

Field trials are the key link to test the effect of DH technology in different environments. For example, a study in Uganda tested 44 newly developed DH test cross hybrids in five locations, focusing on their grain yield and other agronomic traits. The results showed that environmental factors, genotypes and their interactions all significantly affected yield, with the environment explaining nearly half (46.5%) of the variation. It is worth mentioning that the average yield of these DH hybrids was nearly half (49.2%) higher than that of commercial control varieties, showing strong stability and superiority in multiple environments (Sserumaga et al., 2015). This shows that even in complex and changing environments, DH lines still maintain good adaptability.

7.3 Major breeding programs

Globally, several large maize breeding programs have adopted DH technology because it can effectively shorten the breeding cycle and increase the speed of genetic improvement. Rapidly obtaining homozygous lines is very important for these programs, allowing genetic progress to be manifested in a shorter time (Trentin et al., 2020). However, it is worth noting that it is not only large-scale projects that have begun to use DH technology, but also small and medium-scale breeding programs are slowly following suit. Although the promotion speed is relatively slow, in order to adapt to specific environments, especially the needs of tropical regions, these projects are also working hard to cultivate haploid induced lines suitable for local areas. The goal is to make DH technology more accessible and more able to help small breeding operations improve efficiency and genetic benefits.

8 Future Prospects and Research Directions

The application of haploid induction technology in corn breeding has achieved a lot of results, but it still has a lot of room for improvement. One important direction is to improve the haploid induction rate (HIR), which is related to the efficiency and cost of the technology. Recently, it has been found that the maternal HIR can be increased to 16.3% by genetically improving the induced line, such as overexpressing CENH3 in the Stock6 derivative line (Meng et al., 2022). In addition, mutations in the ZmDMP gene can also significantly increase the haploid production rate, sometimes doubling or tripling it (Zhong et al., 2019). Although these advances are promising, there are still many technical details that need to be solved in practical applications.

Although haploid breeding technology has made significant progress, there are still several difficulties that cannot be ignored. For example, how to combine genome editing technology (CRISPR/Cas9) with haploid induction is still in the development stage. The existing haploid induction-mediated genome editing (IMGE) technology can theoretically produce excellent homozygous lines within two generations (Wang et al., 2019), but more optimization is needed for large-scale application. Another challenge is the environmental adaptability of the induced lines, especially in tropical regions, which limits the promotion of the technology (Trentin et al., 2020). Therefore, it is particularly important to develop induced lines that can adapt to different climatic conditions. In addition, the success rate of chromosome doubling is low, and the drugs currently used are toxic. Improvements in this regard are also the focus of future research (Chaikam et al., 2019).

The potential of haploid breeding technology is widely recognized, and its promotion is gradually advancing worldwide. Large-scale corn breeding projects have used this technology as an important tool to quickly obtain homozygous inbred lines and improve the efficiency of genetic improvement (Trentin et al., 2020). However, small and medium-scale breeding programs have made slow progress due to cost and induced line adaptability issues. International cooperation may be able to break this limitation and help more breeding projects benefit by sharing technology and resources. Multinational seed companies have already used DH technology to mass-produce inbred lines of maize hybrids (Dwivedi et al., 2015). If these collaborations can be extended to public institutions and small breeders, the promotion speed will be faster. In addition, collaborative research on the genetic mechanism of haploid induction and the development of new induction lines can also make this technology more popular and practical (Prigge et al., 2012).

Acknowledgments

We would like to thank Professor Luo for her invaluable guidance, insightful suggestions, and continuous support throughout the development of this study.”

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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