A method for improving the induction rate of a maize haploid induction line

Abstract

The present application belongs to the technical field of corn breeding, and particularly relates to a method for improving the induction rate of a corn haploid induction line. The method comprises the step of improving the proportion of medium and low activity pollen in the pollen of the corn haploid induction line, and then pollinating the female parent with the artificially treated pollen of the corn haploid induction line as the male parent to harvest the hybrid contemporary corn haploid seeds. The present application can provide more specific theoretical guidance for the pollination strategy of the corn haploid induction line in production practice, and further improve the efficiency of corn haploid breeding. By means of artificial delayed pollination or in vitro pollination, the proportion of medium and low grade pollen in the pollen of the induction line is improved, and the induction rate of the haploid induction line is further improved.

Description

Technical Field

[0001] This invention belongs to the field of maize breeding technology, specifically relating to a method for improving the induction rate of maize haploid induction lines. Background Technology

[0002] The key to maize hybrid breeding lies in rapidly obtaining pure-line parents with superior and stable traits. Traditional breeding processes involve long selection cycles and low efficiency of inbred lines, making them unsuitable for the intense competition of today's breeding industry. Haploid breeding technology based on haploid inducible lines offers advantages such as low cost, high efficiency, and rapid homozygosity, enabling the rapid acquisition of double haploid (DH) lines and significantly shortening the breeding cycle. Since haploids have only one set of chromosomes, both recessive and dominant genes can be expressed in the current generation. Therefore, screening for superior traits can be performed in early generations, allowing for the timely elimination of undesirable traits. This facilitates the rapid aggregation of beneficial genes such as yield and resistance, improving the efficiency and accuracy of breeding. Maize haploid breeding technology plays an increasingly important role in commercial breeding and, along with molecular breeding and transgenic technology, has become a core technology in modern maize breeding.

[0003] Currently, inducing maize maize maternal haploids in vivo using haploid induction lines derived from the Stock6 maize germplasm is the most widely used method for obtaining maize haploids in production practice. The Stock6 maize haploid induction line was first reported in 1959, at which time the line had a maternal haploid induction rate of only 2.3%-3.2%. However, Stock6 pollen could induce approximately 3% of the maternal material to produce haploid plants. This important discovery laid the theoretical foundation for hybridization-induced haploid breeding methods. In the following decades, breeders worldwide improved the Stock6 induction line through hybridization and backcrossing, selecting several maize maternal haploid induction lines with excellent agronomic traits and significantly improved induction rates (7.0%-16.0%). This led to the successful promotion and application of maize haploid breeding technology based on Stock6 germplasm in commercial maize breeding.

[0004] As of 2016, more than 50 haploid induction lines had been developed globally, all of which are directly or indirectly derived from Stock6. Commonly reported and applied induction lines include RWS, UH400, PK6, and HZI1 from Germany; KMS, ZMS, and KEMS from the Soviet Union; WS14 from France; ZMS and MHI from Moldova; PHI from Romania; 5329C from India; TAILs from CIMMYT; and Nongda Gaoyou 1 and Nongda Gaoyou 5 from China Agricultural University. However, these highly inducible lines often suffer from low seed setting rates and unclear marker traits. In 2013, Chen Shaojiang's research group at China Agricultural University bred an excellent maize haploid induction line, CAU5, using Stock6 germplasm for improvement. In recent years, it has been widely used in commercial maize haploid breeding processes, with a haploid induction rate of approximately 8%-10%, and contains the R1-nj color gene, facilitating haploid identification in the induced offspring kernels. In 2021, China Agricultural University developed a new high-oil haploid induction line, CHOI4, with an average HIR of up to 15.8%. The continuous emergence of new haploid induction lines based on Stock6 has led to a leap forward in reproductive breeding methods for maize that induce haploids, gradually replacing the traditional pedigree breeding method that has been used for over a century.

[0005] In recent years, researchers have conducted in-depth studies on the induction mechanism, successfully identifying the inducing gene and further investigating its functional role. In 2017, Chen Shaojiang's research team discovered that chromosome exclusion during induction is an important mechanism for haploid formation, and identified the phospholipase gene ZmPLA1. The insertion of four bases CGAG in exon 4 of this gene leads to the loss of its original function. The mutated gene abnormally acts on the cell membrane. During pollen tube germination, the abnormality of the cell membrane leads to abnormal gametophyte formation, resulting in a certain proportion of maternal haploid embryos. In 2019, the team further identified the gene ZmDMP, which encodes the DUF679 domain membrane protein, and its functional site. ZmDMP and ZmPLA1 have certain similarities, both being expressed in mature pollen and located on the cell membrane, participating in the same pathway during haploid induction. Based on the ZmPLA1 mutation, a single-base substitution downstream of the ZmDMP start codon leads to amino acid mistranslation, thereby increasing the haploid induction rate by 2-3 times. Complete knockout of this gene, and simultaneous mutation of the mtl / zmpla1 / nld genes, can increase the haploid induction rate by 5-6 times. In recent years, Li et al., in their research on genes in the phospholipase family highly expressed in pollen, discovered a new haploid induction gene, the maize ZmPLD3 haploid induction gene. This gene loss-of-function mutation can induce maternal haploids, with an induction rate basically consistent with ZmPLA1. Compared to the zmpld3 single mutant, the double mutant zmpld3 / mtl of the ZmPLD3 and ZmPLA1 genes can increase the haploid induction rate by 3 times, while the zmpld3 / mtl / zmdmp triple mutant can increase the haploid induction rate by 5-6 times compared to the single mutant.

[0006] Currently, the most common method to improve HIR is still through gene editing and other technologies. This method is time-consuming, costly, and wasteful. Furthermore, the efficiency of improving the induction rate of edited products is unknown, and it may not be suitable for all induction lines. Summary of the Invention

[0007] Technical issues:

[0008] Improving the induction rate of haploid inducible lines is a complex and inefficient process, and has now reached a bottleneck. Even with the successive cloning of inducible genes, simply combining them has not yielded inducible lines with high induction rates. The proportion of haploids produced by traditional pollination methods depends entirely on the induction rate of the inducible line, but we can significantly improve the induction rate by changing the pollination method.

[0009] Developing new induction lines not only requires technical improvements but also wastes human and material resources, has a long cycle, and yields minimal results. Directly optimizing pollination methods can indirectly and rapidly increase the haploid induction rate, maximizing the induction performance of high-induction-rate induction lines.

[0010] Common maize haploid inducible lines, such as the CAU5 inducible line, are tropical germplasm resources. They have low germination rates and high rates of self-pollinated ear abortion, making propagation difficult. Fully utilizing their pollen characteristics to maximize their inducible properties will help to further protect and conserve germplasm resources.

[0011] Therefore, the purpose of this invention is to provide a method for improving the induction rate of maize haploid inducible lines. The method includes the step of increasing the proportion of medium and low viability pollen in the pollen of maize haploid inducible lines, and then using the pollen of maize haploid inducible lines as the male parent to pollinate the female parent, thereby harvesting hybrid contemporary maize haploid kernels.

[0012] Furthermore, methods to increase the proportion of medium and low viability pollen in maize haploid inducible lines include treating maize haploid inducible line pollen with activated carbon;

[0013] The treatment involves storing the pollen and activated carbon separately in a sealed container for 1 to 4 days.

[0014] Furthermore, the storage temperature is 3–5°C.

[0015] Furthermore, the container includes centrifuge tubes with caps.

[0016] Furthermore, the isolated storage involves placing pollen at the bottom of a centrifuge tube and placing activated carbon wrapped in gauze in the middle of the centrifuge tube.

[0017] Furthermore, the treatment was performed during the pollen shedding period of the maize haploid inducible line.

[0018] Furthermore, methods to increase the proportion of medium and low viability pollen in maize haploid inducible lines include delaying pollination;

[0019] The delayed pollination involves isolating the male tassels of the maize haploid inducing line in the field by bagging them for 1 to 4 days, and then using pollen from the maize haploid inducing line as the male parent to pollinate the female parent.

[0020] Furthermore, the period for bagging and isolation coincides with the pollen shedding period of the maize haploid inducible line.

[0021] The present invention has the following beneficial effects:

[0022] The pollination method of the present invention can significantly improve the induction rate of existing maize haploid inducible lines.

[0023] 1) The method provided by this invention can effectively improve the induction rate of existing maize haploid induction lines.

[0024] Existing methods for improving haploid induction rates mainly rely on modifications to inducing genes or genes related to pollen development, with minimal improvement. However, the technique provided by this invention can significantly increase the induction rate of existing inducible lines by simply treating the pollen; it is convenient, easy to implement, and yields substantial improvements.

[0025] 2) The method provided by this invention can be applied to the induction lines of any species and has broad applicability.

[0026] Haploid inducing genes are increasingly being used in various monocotyledonous and dicotyledonous plants, but this is only the initial stage, as simple inducing genes can only provide very low induction rates. The method provided in this invention can be applied to any inducing line variety with a low induction rate, not limited to a specific species. This greatly facilitates the improvement of haploid induction rates in some small crops or vegetable crops, which are limited by self-pollination or the small size and difficulty in identifying seeds.

[0027] 3) The method provided by this invention is simple to operate, low in cost, and has significant effects.

[0028] Compared to traditional methods that use combinations or modifications of inducing genes to increase haploid induction rate, the method provided by this invention is simple, easy to implement, and inexpensive. It can also be used for batch processing during the pollen shedding period without affecting the ripening period of the ears. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the pollen competitiveness experiment (taking a 2-hour interval as an example).

[0030] Figure 2 This is the result of a pollen competitiveness experiment.

[0031] Figure 3 This is a comparison of several pollen storage methods. A: Placed in an uncapped 5ml centrifuge tube; B: Placed in an empty 5ml centrifuge tube with a cap; C: Placed in a sealed 5ml centrifuge tube containing activated charcoal at 4 degrees Celsius, with pollen at the bottom and activated charcoal wrapped in gauze and suspended in the middle of the tube; D: Placed in a 5ml centrifuge tube containing anhydrous CaCl2, with the same setup as C; E: Placed in a 5ml centrifuge tube containing laundry detergent, with the same setup as C; F: Placed in an empty 5ml centrifuge tube placed in an icebox; G: Placed in a sulfuric acid paper bag; H: Placed in a sulfuric acid paper bag containing mixed anther shells; I: Stained image of pollen stored in D, scale bar = 200μm; J: Stained image of pollen stored in C, scale bar = 200μm.

[0032] Figure 4This is a staged pollination experiment using in vitro preserved pollen from the CAU5 inducible line. A: Method and experimental protocol for in vitro preservation of pollen from the maize haploid inducible line CAU5; B: TTC staining results of in vitro preserved pollen from the inducible line CAU5 for 6 consecutive days.

[0033] Figure 5 This is a graph showing the trends in the viability and haploid induction rate of pollen preserved in vitro from the CAU5 induction line.

[0034] Figure 6 This is a field bagging delayed pollination experiment of the CAU5 inducible line. A: Field bagging preservation method and experimental scheme for pollen of the maize haploid inducible line CAU5; B: TTC staining results of pollen of inducible line CAU5 preserved in the field for 6 consecutive days.

[0035] Figure 7 This is a graph showing the changing trends of pollen viability (A) and haploid induction rate (B) of the CAU5 inducible line after field bagging. Detailed Implementation

[0036] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0037] Example 1: Using pollen competitiveness experiments, compare whether there are differences in haploids induced by pollen with different viability.

[0038] A competitive pollination experiment was conducted using pollen from the conventional maize inbred line Qi319 and pollen from the CAU5 or CAUHOI inducible lines. The test variety was Zhengdan 958 (ZD958). Pollen from CAU5 and Qi319 was applied sequentially at different time intervals (0 / 0.5 / 1 / 2 / 4 / 6 hours). The percentage of purple kernels on the ears of the test variety was statistically analyzed (method as follows). Figure 1 (As shown). The results showed that when pollinating Qi 319 pollen preferentially (at intervals of 0.5 hours and 1 hour), no haploids were detected on mature ears in combinations with CAU5 and CAUHOI. This indicates that the pollen of the inducible lines that could compete with and "defeat" Qi 319 pollen (highly viable pollen) ultimately did not induce haploid production. This phenomenon was also verified in the CAUHOI combination.

[0039] If pollination with the inducing line CAU5 is prioritized, from a pollination interval of 0 hours to 6 hours, we will observe that the proportion of purple kernels on mature ears gradually increases. It is only after approximately 2 hours that the proportion of purple kernels on mature ears exceeds 50%. This indicates that prioritizing pollination with the inducing line at 2 hours is necessary to compensate for the fertilization speed deficiency caused by insufficient pollen viability. Simultaneously, the proportion of haploids also gradually increases with the pollination interval, with a subtle increase initially, followed by a significant increase at 4 and 6 hours. Figure 2 Based on the grading of the induced pollen lines, we speculate that the proportion of low-viability pollen and some medium-viability pollen may be the main factors leading to the final formation of haploids, while the proportion of high-viability pollen is relatively small.

[0040] Example 2: Screening of pollen storage methods

[0041] like Figure 3 As shown, on the day of the experiment, corn pollen that was dispersing pollen was collected, and after sifting out the anther shells, it was placed in containers with the following 8 different treatments. Figure 3 In the following cases: A: Placed in an uncapped 5ml centrifuge tube; B: Placed in an empty 5ml centrifuge tube with a cap; C: Placed in a sealed 5ml centrifuge tube containing activated charcoal in a 4°C refrigerator, with pollen at the bottom and activated charcoal wrapped in gauze and suspended in the middle of the tube; D: Placed in a 5ml centrifuge tube containing anhydrous CaCl2; E: Placed in a 5ml centrifuge tube containing laundry detergent; F: Placed in an empty 5ml centrifuge tube placed in an icebox; G: Placed in a sulfuric acid paper bag; H: Placed in a sulfuric acid paper bag containing mixed anther shells.

[0042] The results showed that only method C (5ml centrifuge tubes placed in a 4°C refrigerator with activated carbon) could store pollen well, yielding staining results of J; other treatment methods all produced staining results similar to I. It is worth noting that treatment G resulted in severe pollen clumping, while treatment H improved upon G, and the method of keeping the tubes attached to the tassels also prevented pollen clumping. Therefore, method C was chosen for subsequent experiments.

[0043] Example 3: Comparison of the viability grades and inducibility of pollen stored in vitro for different days.

[0044] Pollen from the inducible line was collected in centrifuge tubes containing activated carbon and stored at 4°C. For six consecutive days starting from the day of collection, a portion of pollen was taken out each day for 2,3,5-triphenyltetrazolium chloride (TTC) staining to identify the proportion of pollen with different viability grades. The results showed that the overall pollen viability of the CAU5 inducible line gradually decreased with increasing storage time. Figure 4 A) The proportion of highly viable and moderately viable pollen gradually decreased, while the proportion of non-viable pollen gradually increased. The proportion of low-viable pollen showed a pattern of first increasing and then decreasing during the 6-day storage period. From day 1 to day 4, the proportion of low-viable pollen rose from 26.9% to 36.5%, then gradually decreased, reaching only 26.4% on day 6. Figure 4 B).

[0045] Next, we studied the distribution of the proportion of medium and low viability pollen. Pollen from the inducing line stored at 4℃ for the above-mentioned number of days was used to pollinate the female ears of the inbred line. The haploid induction rate of the pollinated ears using CAU5 pollen stored in vitro was statistically analyzed. We found that the trend of haploid induction rate showed a pattern consistent with the trend of low viability pollen proportion. Figure 5 A); by Figure 3 It can be seen that the haploid induction rate of ear induced by pollen from in vitro stored inducible lines increased from 7.52% on day 1 to 8.34% on day 4, and then decreased to 5.10% on day 6. Figure 5 B).

[0046] Example 4: Comparison of pollen viability grades and induction ability at different delay times for pollination

[0047] In the field, the male inflorescences of a batch of CAU5 inducible lines were simultaneously covered with pollination parchment bags for isolation. For the next six consecutive days, a portion of the plants were selected daily to collect pollen for TTC staining to assess pollen viability. The proportion of pollen with different viability levels was identified, and the number of days with the highest proportion of medium and low viability pollen was determined. The results showed that the overall viability level of CAU5 pollen in the pollination bags gradually decreased with increasing time between removal and testing. Figure 6 (AB) and the changing trends of pollen at various viability levels over 6 days were basically consistent with those of pollen stored in vitro. Both showed a continuous decrease in the proportion of high and medium viability pollen, especially high viability pollen, whose proportion was almost zero starting on day 4; while the proportion of inactive pollen increased continuously, exceeding 50% after day 4; the proportion of low viability pollen rose from 22.4% on day 1 to 34.7% on day 4, and then decreased to 13.5% on day 6. Compared with CAU5 pollen stored in vitro, although the overall changing trends were consistent, the overall viability level of pollen stored in field bags decreased significantly faster. This is presumably because the field environment is less conducive to pollen preservation, leading to a more rapid decrease in viability compared to that at 4℃. Figure 6 B).

[0048] Furthermore, we determined the conditions under which the proportion of medium and low-grade viability pollen was highest. Pollination of inbred lines with pollen collected from the induction lines over the aforementioned days revealed a similar trend in haploid induction rate as with in vitro stored pollen. Specifically, on days 3 and 4, when the proportion of low-viability pollen was highest, the haploid induction rate reached its highest level over the six days, at 8.06% and 8.11% respectively, significantly higher than the haploid induction rate of fresh CAU5 pollen. Subsequently, as the proportion of non-viability pollen decreased, the haploid induction rate also decreased to 4.86% on day 6. Figure 7 A, B).

[0049] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0050] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0051] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for improving the induction rate of maize haploid inducible lines, characterized in that, The process includes increasing the proportion of low-viability pollen in the pollen of the maize haploid inducible line, then using the maize haploid inducible line pollen as the male parent to pollinate the female parent, and harvesting hybrid contemporary maize haploid kernels. Methods to increase the proportion of low-viability pollen in maize haploid inducible pollen lines include treating maize haploid inducible pollen lines with activated carbon. The treatment involves storing the pollen and activated carbon separately in a sealed container for 4 days at a temperature of 3-5°C. The container is a centrifuge tube with a cap.

2. The method for improving the induction rate of maize haploid induction lines according to claim 1, characterized in that, The isolated storage involves placing pollen at the bottom of a centrifuge tube and placing activated carbon wrapped in gauze in the middle of the centrifuge tube.

3. The method for improving the induction rate of maize haploid induction lines according to claim 2, characterized in that, The treatment was performed during the pollen shedding period of the maize haploid inducible line.

4. The method for improving the induction rate of maize haploid induction lines according to claim 1, characterized in that, Methods to increase the proportion of low-viability pollen in maize haploid inducible lines include delaying pollination; The delayed pollination involves isolating the male tassels of the maize haploid inducible line in the field by bagging them for 4 days, and then using the pollen from the maize haploid inducible line as the male parent to pollinate the female parent.

5. The method for improving the induction rate of maize haploid induction lines according to claim 4, characterized in that, The period for bagging and isolation is the pollen shedding period of the maize haploid inducible line.

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