Systematic production method for improving embryo yield of highland barley-corn hybrid haploid

Abstract

The invention belongs to the field of plant biotechnology and crop genetic breeding, and particularly relates to a systematic production method for increasing the embryo yield of highland barley-corn hybrid haploids. Aiming at the systematic defects of low castration efficiency, large damage, non-specific culture system, blind male parent selection, lack of physiological process regulation and the like in the existing highland barley-corn hybrid induction haploid technology, the invention provides a systematic production method capable of obviously and stably improving the embryo yield of haploid.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology and crop genetics and breeding, specifically relating to a systematic production method for improving the embryo yield of haploid barley-maize hybrids. Background Technology

[0002] Double haploid breeding technology enables heterozygous breeding materials to rapidly become homozygous within a generation, greatly shortening the breeding cycle and making it a key enabling technology in modern agricultural breeding systems. The development and application of haploid breeding technology are particularly active in the breeding of barley and its special type, naked barley, and have mainly formed the following technical pathways:

[0003] 1. Cork Barley Method: This method utilizes the principle that paternal chromosomes are selectively eliminated during embryonic development after hybridization of cultivated barley and cork barley to produce haploids. Several barley varieties and DH lines have been bred using the cork barley method both domestically and internationally. However, this method is affected by many factors, including parental genotypes, cork barley growth conditions, pollination techniques and conditions, and in vitro embryo culture conditions, making it difficult to apply on a large scale to barley haploid breeding.

[0004] 2. Anther / microspore culture: This is a relatively mature and effective technique currently used in barley (highland barley). Chinese research teams have successfully established a microspore regeneration technology system for highland barley and achieved large-scale application, significantly shortening the pure line creation time from 5-8 years to 1-2 years, and cultivating more than 10 new varieties. However, the core bottleneck of this technology lies in its strong genotype dependence; many excellent breeding materials cannot efficiently generate DH lines through this method.

[0005] Inspired by the successful application of wheat × maize technology, inducing haploids in barley using maize pollen is considered a potential universal solution that can theoretically overcome the genotypic limitations of all the above methods.

[0006] However, research on the application of this technology in highland barley is severely lagging behind, and its industrialization faces multiple systemic bottlenecks:

[0007] Issues with emasculation efficiency and quality: Barley is a typical cleistogamous pollinator, with its flowers tightly enclosed by the glume. Traditional emasculation with tweezers results in a narrow operating space, extremely low efficiency (approximately 8-12 spikes / person / hour), and is prone to causing mechanical damage to the delicate stigma and ovary. Furthermore, the excessively long processing time increases the risk of rachis breakage, leading to a severe shortage of usable florets for hybridization.

[0008] The in vitro culture support system is not specific: Currently, there is a lack of dedicated in vitro culture programs for the physiological characteristics of barley. Existing technologies mostly borrow from wheat formulations, and their single-stage culture media fail to optimize for the characteristics of barley, such as easy browning after in vitro culture and high requirements for calcium and antioxidants. They also do not consider the key requirement of physiological synchronization of the ear before pollination, resulting in poor ear activity and insufficient support for embryo initiation signals.

[0009] The induction efficiency of maize paternal parents is random and low: different maize genotypes exhibit significant differences in their ability to induce haploids in barley, but in production, there is a lack of scientifically screened, stable, and highly efficient dedicated maize paternal parents. The arbitrariness in paternal parent selection leads to large fluctuations in embryo yield, making it difficult to predict and improve, thus becoming a core factor restricting the stable application of the technology.

[0010] The regulation of physiological processes is crude: existing methods usually regard emasculation, pollination and culture as isolated steps, lacking the link of actively regulating the physiological state of barley detached ears (especially the developmental synchronicity of florets and stigma activity), resulting in a low match between pollination timing and the optimal pollination period of florets, and limited effective fertilization rate.

[0011] In summary, a significant technological gap exists in the current field of haploid breeding of highland barley: on the one hand, mature microspore technology is limited by genotype; on the other hand, the theoretically broad-spectrum highland barley × maize technology is hampered by inefficient, unstable, and unsystematic operational systems, and its enormous potential remains largely untapped. Therefore, the industry urgently needs a haploid-induced production method for highland barley × maize that is independent of specific highland barley genotypes and involves systematic optimization across the entire chain from source to output. This method would overcome efficiency bottlenecks, enabling the large-scale, industrialized application of this technology and providing a new, efficient, and universally applicable pathway for creating DH lines in highland barley breeding. Summary of the Invention

[0012] To address the above problems, this invention provides a systematic production method for improving the embryo yield of haploid barley-maize hybrids, comprising the following steps:

[0013] (1) The barley mother ears that were in the 1-2 cm awn-out stage before the husks broke were treated by the hull-cutting and emasculation method to obtain detached ears;

[0014] (2) Place the isolated spikelet in the pre-culture solution and pre-culture for 40-52 hours;

[0015] (3) After pre-culture, pollen from the maize variety 'Yunyoutian 2' was used for centralized pollination;

[0016] (4) Immediately after pollination, place the spikelets in an initial culture medium containing 90-110 mg / L 2,4-D and 90-110 mg / L gibberellin GA3 and culture them under the same conditions for 12-14 days;

[0017] (5) After completing step (4), perform subsequent embryo rescue.

[0018] Further, the pre-culture solution in step (2) comprises per liter of potassium dihydrogen phosphate 2g, urea 1.5g, sucrose 20g, silver nitrate 0.05g, sulfurous acid 4ml, Hogland nutrient solution dry powder 463.8mg, and 1ml of 1000× calcium concentrate; the working concentration of the Hogland nutrient solution dry powder per liter provides the following components: KH2PO4 27.218mg, MgSO4 120.36mg, KCl 111.825mg, CaCl2 166.47mg, H3BO3 0.6183mg, MnSO4·H2O 0.1690mg, ZnSO4·7H2O 0.2875mg, CuSO4·5H2O 0.1248mg, FeNaEDTA 36.705mg, (NH4)6Mo7O 24 ·4H2O 0.0618mg; the 1000× calcium concentrate is an aqueous solution of Ca(NO3)2·4H2O, which provides 472.3mg of Ca(NO3)2·4H2O per liter of working concentration.

[0019] Furthermore, the pre-culture time in step (2) is 48 hours, and the light intensity is 2000 LUX.

[0020] Furthermore, the pre-culture temperature in step (2) is 22-24℃ and the humidity is 85-95%.

[0021] Furthermore, in step (4), the concentrations of 2,4-D and gibberellin GA3 in the initial culture medium are both 100 mg / L, and the culture time is 12-14 days.

[0022] Furthermore, the subsequent embryo rescue and acquisition described in step (5) specifically includes: after completing the initial 24-hour culture of pollination, removing the ears of wheat from the culture medium and spraying a mixed solution of 2,4-D and gibberellin GA3 onto the ears of wheat, wherein the concentration of 2,4-D and GA3 in the mixed solution is 50 mg / L; after spraying, air-drying and continuing culture for 12-14 days, and then peeling off the grains to rescue the embryos.

[0023] This invention also provides the application of the maize variety 'Yunyoutian 2' in improving the embryo yield of haploid barley-maize hybrids.

[0024] The present invention has the following beneficial effects:

[0025] 1. Order-of-magnitude improvement in embryo yield: Through system optimization, the method of this invention can stably increase the embryo yield of haploid barley-maize hybrids to 14%-17% (using 'Yunyoutian 2' as the male parent). Compared with the use of traditional methods and inefficient male parents (embryo yield is often below 5% or even 0%), this method achieves several times the increase and solves the core bottleneck of yield.

[0026] 2. Overcame the global challenge of emasculation in barley: the "emasculation removal method" increases efficiency by 3-4 times, minimizes damage, makes large-scale hybridization possible, and breaks through the limitations of manpower and materials for large-scale production.

[0027] 3. An innovative physiological regulation dimension has been introduced: the "active synchronous pre-culture" process actively regulates the physiological state of the florets to the optimal pollination period, maximizing the utilization of the effective pollination window. This is an important scientific supplement and optimization to the traditional process.

[0028] 4. A data-based parent selection standard was established: Through systematic screening, efficient and dedicated parent bases such as 'Yunyoutian 2' were identified, transforming parent base selection from empiricism to scientific decision-making, thus ensuring the stability and repeatability of the technical effects.

[0029] This invention forms an end-to-end standardized production line: It provides a complete, clear, and operable standard operating procedure from field sampling and laboratory processing to final output. The technologies at each stage work synergistically, making it easy to replicate and promote in different breeding units, and possessing strong potential for industrial application. Figure 1 ). Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a complete process flow diagram of the systematic production method of the present invention.

[0032] Figure 2 A bar chart comparing the embryo yield rates of haploids induced from different maize varieties 'Zangqing 3000' and 'Yunqing 604' (based on data in Table 1).

[0033] Figure 3Figure A shows a comparison of the state of barley plants and ears, as well as the size and fullness of caryopsis, after cultivation using the culture solution B of this invention and a traditional culture solution. Figure B compares the state of barley plants after 14 days of cultivation using the culture solution B of this invention and a traditional wheat culture solution; Figure C compares the state of barley after 14 days of cultivation using the culture solution B of this invention and a traditional wheat culture solution. Detailed Implementation

[0034] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] Example 1: Comparative experiment of the "emasculation and pre-cultivation" method of the present invention and the traditional method in terms of emasculation efficiency and fruit yield.

[0042] This embodiment aims to quantitatively verify, through rigorous comparative experiments, the significant advantages of the "emasculation and emasculation method" combined with the "active synchronous pre-culture" step proposed in this invention compared to traditional methods.

[0043] I. Materials and Methods

[0044] 1. Test materials: Five representative highland barley varieties were selected: 'Zangqing 3000 (six-ridged)', 'T28 (six-ridged)', 'T78 (six-ridged)', 'Yunqing 602 (two-ridged)', and 'Yunqing 604 (two-ridged)'.

[0045] 2. Experimental Design: Each variety was replicated three times. Two treatment groups were set up:

[0046] Control group (traditional method): Traditional tweezers demasking method was used. In the field, the anthers of each female ear were removed one by one with tweezers, and the ears were then bagged for isolation. Two days later, when the stigmas of the naturally growing ears in the field reached their optimal state of feathering, the ears were cut off for corn pollination.

[0047] Experimental group (method of this invention): The "emasculation method" described in this invention was used. Emasculation was performed by directly cutting the anthers in the field, and the ears of wheat were immediately cut from the base of the plant. The detached ears were inserted into the special pre-culture solution (solution A) of this invention and cultured in an artificial climate chamber (23±1°C, 90% humidity, 2000 LUX light, 14h / 10h light / dark cycle) for 2 days (48 hours). After the culture was completed, when all the stigmas of the ears reached the optimal feathering state simultaneously, maize pollination was carried out.

[0048] 2. Key Operations and Statistics: Record the efficiency of emasculation for each replicate (spike / (person·hour)). After pollination, both groups were cultured under identical culture medium and conditions for 14 days. After culture, the total number of florets and the number of caryopsis developed per spike were counted, and the caryopsis percentage (number of caryopsis / number of florets × 100%) was calculated.

[0049] 3. Pollination and cultivation: To ensure comparability, both groups used the same batch of 'Yunyoutian 2' corn pollen, and the hormone treatment, culture medium and culture environment after pollination were exactly the same.

[0050] II. Results and Analysis in conclusion

[0051] 1. Revolutionary breakthrough in detasseling efficiency: Among all five barley varieties tested, the detasseling efficiency of the method of this invention (28-34 ears / person·hour) was significantly higher than that of the traditional method (5-14 ears / person·hour), with an average efficiency improvement of about 3-5 times. This fundamentally solves the human resource bottleneck in the large-scale production of barley double haploid breeding.

[0052] 2. Systematic and significant improvement in fruit yield: The average fruit yield obtained by the method of this invention is consistently above 90% (87.25%-98.60%), while the fruit yield of traditional methods fluctuates greatly and is generally lower (64.71%-79.46%). This indicates that the "in vitro pre-culture" step of this invention can more effectively and synchronously activate and maintain stigma activity, thereby significantly improving pollination success rate and fertilization efficiency.

[0053] 3. Stable and universally applicable: The method of this invention has shown high efficiency and stability for different genotypes of barley varieties (including different ridge shapes), proving the broad applicability and reliability of the technology system.

[0054] This embodiment fully demonstrates that the "pruning and emasculation-pre-culture" system provided by the present invention not only has a revolutionary advantage in operational efficiency, but more importantly, it significantly improves the utilization efficiency of reproductive organs through active regulation of physiological processes, laying a solid material foundation for obtaining a high embryo yield in the future.

[0055] Example 2: Comparative experiment of haploid embryo yield rate between the whole system of the present invention (special culture medium B) and the control system (wheat culture medium).

[0056] This embodiment aims to verify that, after unifying and optimizing the front-end emasculation and pollination process, the barley-specific initial post-pollination culture medium (Liquid B) and subsequent treatment system designed in this invention play a key role in improving the haploid embryo yield rate compared to directly using the existing wheat patent culture medium.

[0057] I. Materials and Methods

[0058] 1. Test materials: Five highland barley varieties were selected: 'Zangqing 3000', 'T28', 'T78', 'Yunqing 602', and 'Yunqing 604'. Pollen from the high-induction maize variety 'Yunyoutian 2' was used uniformly.

[0059] 2. Experimental Design: Control Group (Traditional Culture System): Stamens were removed using traditional forceps, followed by bagging. Two days later, when the stigmas matured, the spikelets were cut and pollinated with 'Yunyoutian 2' pollen. After pollination, the entire culture was performed using the in vitro culture medium (formula: potassium dihydrogen phosphate 1.25 g / L, urea 1.5 g / L, boric acid 100 mg / L, citric acid 20 mg / L, 2,4-D 50 mg / L, sucrose 20 g / L) as described in Chinese Patent (Wheat Patent) with publication number CN113526999A, and hormone treatment was performed according to its recommended methods.

[0060] Experimental group (the entire system of this invention): The entire process described in claim 1 of this invention is adopted. That is: emasculation and removal of male flowers → insertion into pre-culture solution A, pre-cultured in a growth chamber (23±1°C, 90% humidity, 2000 LUX) for 2 days → pollination with 'Yunyoutian 2' pollen → immediately after pollination, transfer to the special initial culture solution B of this invention for 24 hours → spray with a 50 mg / L mixed solution of 2,4-D and GA3 → continue culturing until day 14.

[0061] 3. Observation and Statistics: Each group was set up with 3 replicates, and the number of spikelets and the total number of florets in each replicate were recorded. After the culture was completed, the number of developed caryopsis fruits was counted, and the caryopsis rate was calculated. Subsequently, the number of haploid embryos in all caryopsis fruits was removed by microscopic examination, and the embryo yield rate (number of embryos / number of pollinated florets × 100%) was calculated.

[0062] 4. II. Results and Analysis III. Conclusion

[0063] Embryo yield is increased by an order of magnitude: In all five barley varieties tested, the average haploid embryo yield of the entire system (experimental group) using this invention remained stable between 12% and 20% (specific range: 11.41%-20.58%). In contrast, the embryo yield of the traditional wheat culture medium system (control group) was generally below 2.5%, with multiple replicates even below 1%. This invention increases the embryo yield by at least one order of magnitude (5 to tens of times), demonstrating extremely significant results.

[0064] Synergistic optimization of caryopsis rate and embryo yield: The high embryo yield in the experimental group was based on an extremely high and stable caryopsis rate (85%-95%), which indicates that the "pre-culture" and "special B solution" of this invention not only promoted fertilization and caryopsis development, but more importantly, provided key support for the successful initiation and development of subsequent embryos. Figure 3 The low lemma rate in the control group directly limited the upper limit of the number of embryos that could be obtained.

[0065] The system exhibits significant synergistic effects and strong universality: This invention is not merely an improvement on a single formula. The high yield in the experimental group is the result of the synergistic effect of the entire system: "efficient emasculation → synchronized pre-culture → highly inducible paternal parent → specialized high-hormone initiation culture medium." Data shows that this system demonstrates efficient and stable induction ability for barley varieties with different genetic backgrounds (Zangqing 3000, T series, and Yunqing series), proving its reliability and industrialization potential as a universal solution.

[0066] Creativity highlighted: This embodiment directly demonstrates that applying the culture medium from the wheat patent to barley, even with a highly inducible parent, still results in an extremely low embryo yield. This proves that the dedicated two-stage culture medium system (especially solution B) developed for barley characteristics in this invention is not an obvious, simple replacement, but a key technological innovation with outstanding and substantial characteristics that solves the problem of haploid induction specificity in barley.

[0067] This embodiment comprehensively demonstrates that the systematic production method provided by the present invention, through the interconnected integration of technologies, achieves a fundamental breakthrough in the "efficiency bottleneck" and "output bottleneck" in the induction of haploid embryos in barley-maize hybrids, and can stably and efficiently produce haploid embryos of barley, thus possessing significant breeding application value.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A systematic production method for improving the embryo yield of haploid barley-maize hybrids, characterized in that, Includes the following steps: (1) The barley mother ears that were in the 1-2 cm awn-out stage before the husks broke were treated by the hull-cutting and emasculation method to obtain detached ears; (2) Place the isolated spikelet in the pre-culture solution and pre-culture for 40-52 hours; (3) After pre-culture, pollen from the maize variety 'Yunyoutian 2' was used for centralized pollination; (4) Immediately after pollination, place the spikelets in an initial culture medium containing 90-110 mg / L 2,4-D and 90-110 mg / L gibberellin GA3 and culture them under the same conditions for 12-14 days; (5) After completing step (4), perform subsequent embryo rescue.

2. The production method according to claim 1, characterized in that, The pre-culture solution in step (2) comprises per liter of potassium dihydrogen phosphate 2g, urea 1.5g, sucrose 20g, silver nitrate 0.05g, sulfurous acid 4ml, Hogland nutrient solution dry powder 463.8mg, and 1ml of 1000× calcium concentrate; the working concentration of the Hogland nutrient solution dry powder per liter provides the following components: KH2PO4 27.218mg, MgSO4 120.36mg, KCl 111.825mg, CaCl2 166.47mg, H3BO3 0.6183mg, MnSO4·H2O 0.1690mg, ZnSO4·7H2O 0.2875mg, CuSO4·5H2O 0.1248mg, FeNaEDTA 36.705mg, (NH4)6Mo7O 24 ·4H2O 0.0618mg; the 1000× calcium concentrate is an aqueous solution of Ca(NO3)2·4H2O, which provides 472.3mg of Ca(NO3)2·4H2O per liter of working concentration.

3. The production method according to claim 1, characterized in that, The pre-culture time in step (2) is 48 hours, and the light intensity is 2000 LUX.

4. The production method according to claim 1, characterized in that, The pre-culture temperature in step (2) is 22-24℃ and the humidity is 85-95%.

5. The production method according to claim 1, characterized in that, In step (4), the concentrations of 2,4-D and gibberellin GA3 in the initial culture medium are both 100 mg / L, and the culture time is 12-14 days.

6. The production method according to claim 1, characterized in that, The subsequent embryo rescue and acquisition described in step (5) specifically includes: after completing the initial 24-hour culture of pollination, the ears of wheat are removed from the culture medium and a mixed solution of 2,4-D and gibberellin GA3 is sprayed onto the ears of wheat. The concentration of 2,4-D and GA3 in the mixed solution is 50 mg / L. After spraying, the ears are air-dried and cultured for another 12-14 days. Then, the ears of wheat are peeled off to rescue the embryos.

7. Application of maize variety 'Yunyoutian 2' in improving the embryo yield of haploid barley-maize hybrids.

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