Semi-winter and winter wheat rapid homozygosis method based on haploid induction line

Abstract

The invention provides a rapid homozygosis method for semi-winter and winter wheat based on a haploid induction line, and belongs to the technical field of plant breeding. Comprising the following steps: (1) taking a purple embryo induction line PEI as a male parent and taking hybrid semi-winter or hybrid winter wheat as a female parent, and hybridizing to obtain hybrid seeds; (2) selecting seeds with normal embryo color from the hybrid seeds as haploid seeds; (3) accelerating germination and vernalization of the haploid seeds, and then performing seedling culture; (4) after the seedlings are cultured to a four-to-five-leaf stage, taking the seedlings out of the nutrient soil, cleaning roots, and immersing tillering joints and the roots of the seedlings into a solution containing colchicine, DMSO and GA3 for doubling treatment; and (5) transplanting and culturing the doubled seedlings to promote the formation of new tillers, and obtaining homozygous double haploid seeds after 50-70 days. The technology provided by the invention improves the wheat breeding efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of plant breeding, and in particular to a rapid homozygous method for semi-winter and winter wheat based on a haploid induction line. Background Art

[0002] For a long time, in order to ensure the consistency of varieties, the wheat varieties promoted in production have been obtained through years of self-pollination or backcrossing to obtain stable pure lines. However, the acquisition of pure lines is the longest cycle and the most expensive link in the entire breeding process. If the creation process of pure lines can be accelerated, the efficiency of wheat breeding can be significantly improved.

[0003] Double haploid breeding is an effective method for rapid pure line breeding in production. In the 1970s, Chinese scientists made a major breakthrough in the in vitro culture of wheat anthers, but this technology is highly dependent on genotypes; in the 1980s, Laurie et al. obtained wheat haploids by using wheat × corn distant hybridization. To date, many units in my country are still using corn pollen to induce haploid formation in wheat to improve breeding efficiency. However, there are some problems in the actual application process. On the one hand, corn pollen cannot be stored for a long time, and it is necessary to sow corn in stages according to the flowering time of wheat so that their flowering periods meet. Corn growth requires a large amount of light and temperature resources, and off-season operations require the help of light and temperature equipment; on the other hand, the embryoids obtained by pollinating wheat with corn need to be differentiated into haploid seedlings with the help of tissue culture technology. The operation process is relatively cumbersome and easy to be infected with bacteria, resulting in haploid loss; third, the process of making haploid embryos into seedlings through tissue culture cannot be interrupted, and the experimental conditions for continuous planting of wheat are required. In summary, the wheat haploid breeding method based on interspecific hybridization has many limitations, and new haploid breeding technologies need to be explored to improve wheat breeding efficiency.

[0004] Recently, wheat haploids were induced by knocking out wheat TaPLA / TaMTL genes through gene editing technology (Liu HY, Wang K, Jia ZM, et al. Editing TaMTL gene induces haploid plants efficiently by optimized Agrobacterium-mediated CRISPR system in wheat. J Exp Bot, 2020a, 71(4): 1337-1349.). The TaPLA mutant was used as the father and hybridized with the target parent, and 5-30% haploid seeds were obtained in the offspring (Liu CX, Zhong Y, Qi XL, et al. Extension of the in vivo haploid induction system from diploid maize to hexaploid wheat. Plant Biotechnol J, 2020b, 18(2): 316-318.). Afterwards, in order to efficiently and accurately identify haploids from hybrid grains, Qi et al. transformed the embryo-specific promoter-driven anthocyanin synthesis regulatory genes ZmC1 and ZmR expression vectors into the TaPLA mutant, and obtained the haploid induction line PEI with color screening markers (Qi X, Guo S, Zhong Y, Chen B, Liu Z, Yan T, Feng B, Ni Z, Chen S, Liu C. Establishment of an efficient haploid identification system by engineering anthocyanin accumulation in the wheat embryo. Plant Communications. 2023 May 8; 4 (3).). Therefore, the present invention studies a rapid breeding method that does not rely on tissue culture and is not restricted by wheat genotype and is simple to operate for semi-winter and winter wheat, so as to improve the efficiency of wheat breeding. Summary of the invention

[0005] The purpose of the present invention is to provide a rapid homozygous technology for semi-winter and winter wheat based on a haploid induction line, so as to improve the efficiency of wheat breeding.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for rapid homozygosity of semi-winter and winter wheat based on a haploid induction line, comprising the following steps:

[0008] (1) using the purple embryo induction line PEI as the male parent and the heterozygous semi-winter or heterozygous winter wheat as the female parent, hybridizing to obtain hybrid seeds;

[0009] (2) selecting normal embryo color seeds as haploid seeds among the hybrid seeds;

[0010] (3) germinating and vernalizing the haploid seeds, and then culturing seedlings;

[0011] (4) When the seedlings are cultured to the four- to five-leaf stage, the seedlings are taken out of the nutrient soil, the roots are cleaned, and the tillering nodes and roots of the seedlings are immersed in a solution containing colchicine, DMSO, and GA3 for doubling treatment;

[0012] (5) Transplanting and culturing the doubling treated seedlings to promote the formation of new tillers, and obtaining homozygous double haploid seeds after 50 to 70 days.

[0013] Preferably, the purple embryo induction line PEI in step (1) is a TaPLA gene-edited strain and anthocyanin synthesis regulatory factors are introduced, and the background is spring wheat; the maternal parent is any semi-winter or winter wheat whose genome is in a heterozygous state.

[0014] Preferably, the germination in step (3) is carried out by treating the seeds with 1.2% H2O2 at 22-25°C for 40-48h; the ungerminated seeds are continued to be placed in a 1.2% H2O2 solution at 4-8°C until they germinate.

[0015] Preferably, the vernalization process in step (3) is: placing the germinated seeds in a refrigerator at 4 to 8° C. for 30 to 35 days.

[0016] Preferably, the seedling cultivation process of step (3) is as follows: the seedlings after vernalization are transplanted into a hole tray containing nutrient soil, and cultured in alternating light and dark conditions, wherein the light duration is 16 hours, the light intensity is 2800-3200Lx; the dark duration is 8 hours; the temperature is 25-28°C, until the seedlings grow to 4-5 leaves.

[0017] Preferably, in step (4), the concentration of colchicine is 0.4 g / L, the concentration of DMSO is 20 ml / L, the concentration of GA3 is 100 mg / L, the doubling treatment time is 8 h, the treatment temperature is 20-22° C., and air flow is slowly introduced during the treatment at a flow rate of 1 L / min.

[0018] Preferably, the cultivation in step (5) is carried out at 16-18°C with alternating light and dark for 10-14 days, during which the light time is 15-17 hours, the light intensity is 2800-3200Lx, and the dark time is 9-7 hours; then the temperature is adjusted to 24-26°C and the light-dark alternating cultivation is continued, and homozygous double haploid seeds are harvested after 50-70 days.

[0019] The present invention provides a rapid homozygous technology for semi-winter and winter wheat based on a haploid induction line. The technology uses a purple embryo induction line PEI with anthocyanin screening markers as the male parent and a heterozygous semi-winter and winter wheat as the female parent, and the two are hybridized; haploid screening is performed according to the anthocyanin accumulation in the offspring embryo; after obtaining haploid seeds, double haploid seeds can be obtained within 100 to 120 days. Compared with the process of anther in vitro culture and corn pollen induction of double haploid wheat, the technology does not rely on tissue culture and is easy to operate; at the same time, the method has low dependence on wheat genotype; and the process can be implemented in any wheat planting area without geographical restrictions. DETAILED DESCRIPTION

[0020] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0021] Example 1

[0022] The semi-winter wheat Xumai DH9 was hybridized with the semi-winter wheat Xumai 45 to obtain the F1 generation hybrid wheat, which was sown in the experimental field on October 8, 2023; the haploid induction line PEI with anthocyanin selection markers was sown in three phases on November 1, November 8 and November 15, 2023; the PEI induction line was a TaPLA gene-edited strain and an embryo-specific expression vector-driven anthocyanin synthesis regulatory factor was introduced, and the background was spring wheat Fielder (Qi X, Guo S, Zhong Y, Chen B, Liu Z, Yan T, Feng B, Ni Z, Chen S, Liu C. Establishment of an efficient haploid identification system by engineering anthocyanin accumulation in the wheat embryo. Plant Communications. 2023May 8; 4(3).). On December 30, 2023, PEI was covered with a film for insulation, and on February 15 of the following year, the film was removed and allowed to grow naturally.

[0023] The whole ears of F1 generation heterozygous semi-winter wheat were artificially emasculated 2 days after heading, with a total of 15 ears emasculated and bagged for isolation; 3 days after emasculation, fresh induced PEI pollen was applied to the emasculated wheat stigmas, which were then bagged for isolation.

[0024] After the hybrid seeds matured, the haploid seeds were identified according to the haploid identification method (CN114946639A), that is, the purple embryo seeds were diploid because they carried the PEI chromosomes; the haploid seeds did not contain the PEI chromosomes, and the embryos were normal in color; there were also some embryoless seeds in the offspring. A total of 42 purple embryo seeds, 25 embryoless seeds, and 24 normal embryo color seeds were obtained in this hybridization.

[0025] 24 haploid seeds with normal embryo color were selected and soaked in 1.2% H2O2 at 24℃ for 48h. 22 seeds turned white. They were washed with clean water and placed in a 5℃ refrigerator for vernalization. The 2 seeds that did not germinate were further soaked in 1.2% H2O2 solution and also transferred to a 5℃ refrigerator. After 3 days, the 2 seeds germinated. The H2O2 solution remaining on the surface was washed with clean water and continued to be vernalized in a 5℃ refrigerator. Vernalization ended after 30d.

[0026] Transplant the seedlings into a hole tray containing nutrient soil and carry out alternating light and dark cultivation, wherein the light time is 16 hours, the light intensity is 2800Lx; the dark time is 8 hours; the temperature is 25-28°C; until the seedlings grow to the 4-5 leaf stage.

[0027] When the seedlings grow to the stage of 4 to 5 leaves, remove the seedlings from the hole tray and wash the nutrient soil around the roots.

[0028] The seedlings were bundled, and the tiller nodes and roots were immersed in a solution containing 0.4 g / l colchicine, 18 ml / l DMSO and 100 mg / l GA3 for 8 hours at a temperature of 22°C. During this period, air was slowly introduced at a flow rate of 1 L / min.

[0029] Take out the seedlings and rinse them with slow-flowing water overnight. The next day, transplant the seedlings into pots with appropriate slow-release fertilizer and place them in an environment of 18°C, 16h light, 2800Lx light intensity, and 8h dark for 12 days to promote the formation of new tillers. Then adjust the temperature to 26°C, 16h light, 2800Lx light intensity, and 8h dark for cultivation.

[0030] After the above treatment, 5 of the 24 haploid seedlings died, and the rest doubled successfully, and double haploid seeds were harvested. The growth cycle was 58 days.

[0031] Example 2

[0032] The semi-winter wheat Xumai DH9 was hybridized with the winter wheat variety Nongda 3432, and the obtained F1 hybrid seeds were sown in the field on October 8, 2023; the haploid induction line PEI with anthocyanin selection markers was sown on November 1, November 8 and November 15, 2023, respectively.

[0033] After the F1 generation of heterozygous wheat had headed for 2 days, the whole ear was artificially emasculated, and a total of 15 ears were emasculated and bagged for isolation; 3 days after emasculation, fresh induced PEI pollen was applied to the emasculated wheat stigma, which was then bagged for isolation. After maturity, the grains were harvested, and a total of 44 purple embryo seeds, 20 normal embryo seeds, and 26 embryoless seeds were harvested.

[0034] 20 haploid seeds with normal embryo color were selected and soaked in 1.2% H2O2 at 24℃ for 48h. 18 seeds turned white and were washed with clean water and placed in a 5℃ refrigerator for vernalization. 2 seeds that did not germinate were further soaked in 1.2% H2O2 solution and also transferred to a 5℃ refrigerator. After 4 days, 2 seeds germinated and the residual H2O2 solution on the surface was washed with clean water and continued to be vernalized in a 5℃ refrigerator. Vernalization ended after 35d.

[0035] Transplant the seedlings into a hole tray containing nutrient soil and carry out alternating light and dark cultivation, wherein the light time is 16 hours, the light intensity is 2800Lx; the dark time is 8 hours; the temperature is 25-28°C; until the seedlings grow to the 4-5 leaf stage.

[0036] When the seedlings grow to the stage of 4 to 5 leaves, remove the seedlings from the hole tray and wash the nutrient soil around the roots.

[0037] The seedlings were bundled, and the tiller nodes and roots were immersed in a solution containing 0.4 g / l colchicine, 18 ml / l DMSO and 100 mg / l GA3 for 8 hours at a temperature of 22°C. During this period, air was slowly introduced at a flow rate of 1 L / min.

[0038] Take out the seedlings and rinse them with slow-flowing water overnight. The next day, transplant the seedlings into pots with appropriate slow-release fertilizer and place them in an environment of 18°C, 16h light, 2800Lx light intensity, and 8h dark for 12 days to promote the formation of new tillers. Then adjust the temperature to 26°C, 16h light, 2800Lx light intensity, and 8h dark for cultivation.

[0039] After the above treatment, 4 of the 20 haploid seedlings died, and the rest doubled successfully, and double haploid seeds were harvested. The growth cycle was 66 days.

[0040] The invention takes about 100 to 120 days from the harvesting of haploid seeds to the obtaining of double haploid seeds. If the haploid seeds are harvested in time in summer, the double haploid seeds are harvested in early to mid-October, which is the suitable sowing period for semi-winter or winter wheat, and can be sown and propagated in the field.

[0041] Compared with the process of anther in vitro culture and corn pollen induction of wheat double haploid, the breeding process of the present invention has obvious advantages: first, it does not involve tissue culture and is easy to operate; second, the method is not limited by wheat genotype and is suitable for most semi-winter and winter wheat; and the process can be implemented in any wheat-growing area without geographical restrictions.

[0042] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for rapid homozygosity of semi-winter and winter wheat based on haploid induction lines, characterized in that: The steps include: (1) using the purple embryo induction line PEI as the male parent and the heterozygous semi-winter or heterozygous winter wheat as the female parent, hybridizing to obtain hybrid seeds; (2) selecting normal embryo color seeds as haploid seeds among the hybrid seeds; (3) germinating and vernalizing the haploid seeds, and then culturing seedlings; (4) When the seedlings are cultured to the four- to five-leaf stage, the seedlings are taken out of the nutrient soil, the roots are cleaned, and the tillering nodes and roots of the seedlings are immersed in a solution containing colchicine, DMSO, and GA3 for doubling treatment; (5) Transplanting and culturing the doubling treated seedlings to promote the formation of new tillers, and obtaining homozygous double haploid seeds after 50 to 70 days.

2. The method according to claim 1, characterized in that The purple embryo induction line PEI in step (1) is a TaPLA gene-edited strain and anthocyanin synthesis regulatory factors have been introduced, and the background is spring wheat; the maternal parent is any winter or semi-winter wheat whose genome is in a heterozygous state.

3. The method according to claim 2, characterized in that The germination in step (3) is carried out by treating the seeds with 1.2% H2O2 at 22-25°C for 40-48h; the ungerminated seeds are further placed in the 1.2% H2O2 solution at 4-8°C until they germinate.

4. The method according to claim 3, characterized in that The vernalization process in step (3) is as follows: placing the germinated seeds in a refrigerator at 4 to 8° C. for 30 to 35 days.

5. The method according to claim 4, characterized in that The seedling cultivation process of step (3) is as follows: the seedlings after vernalization are transplanted into a hole tray containing nutrient soil and cultured in alternating light and dark conditions, wherein the light duration is 14-18 hours, the light intensity is 2800-3200Lx; the dark duration is 6-10 hours; the temperature is 25-28°C, until the seedlings grow to 4-5 leaves.

6. The method according to claim 5, characterized in that In step (4), the concentration of colchicine is 0.4 g / L, the concentration of DMSO is 20 ml / L, the concentration of GA3 is 100 mg / L, the doubling treatment time is 8 h, the treatment temperature is 20-22° C., and air flow is slowly introduced during the treatment at a flow rate of 1 L / min.

7. The method according to claim 6, characterized in that The cultivation in step (5) is carried out under the condition of 16-18°C with alternating light and dark for 10-14 days. During the alternating light and dark cultivation, the illumination time is 14-16 hours, the illumination intensity is 2800-3200Lx; the dark time is 8-10 hours; then the temperature is adjusted to 24-26° and the alternating light and dark cultivation is continued. After 50-60 days, the homozygous double haploid seeds are harvested.