A method for quickly creating new germplasm of late bolting and downy mildew resistant radish

Abstract

The application discloses a method for quickly creating new radish germplasm with late bolting and downy mildew resistance, and belongs to the technical field of crop breeding. The method comprises the following steps: obtaining F1 by crossing radish downy mildew resistant material DM2 and bolting resistant material LB; screening target trait plants after seed vernalization; taking suitable flower buds for 4 DEG C pretreatment for 24 hours; suspending the isolated and purified microspores in 1 / 2NLN medium added with 1.0 muM NaB; obtaining regenerated plants through differentiation, bud strengthening and rooting culture; transplanting the seedlings after hardening and vernalization; screening double haploid single plants with genotype homozygosity and both late bolting and downy mildew resistance by using InDel markers and genome resequencing in combination with field agronomic trait identification; and obtaining new germplasm by selfing and seed saving. The application uses polymerization breeding and microspore culture technology to quickly create excellent radish germplasm with complete genome homozygosity, late bolting and downy mildew resistance, and provides important technical support for accelerating genetic improvement of radish late bolting, high quality and multi-resistance varieties.

Description

Technical Field

[0001] This invention belongs to the field of crop bio-breeding technology, specifically relating to a method for rapidly creating new radish germplasm that is late-bolting and resistant to downy mildew. Background Technology

[0002] radish( Raphanus sativus L . Radish (Raphanus sativus) is an annual or biennial root vegetable belonging to the Brassicaceae family and the Raphanus genus. It is a typical seed-vernalizing vegetable and is widely cultivated worldwide. Radishes are characterized by rapid growth, high yield, strong resistance to adverse conditions, and good storage and transportation qualities. However, premature bolting is one of the core issues affecting the quality and marketability of radishes during winter and spring production. After low-temperature vernalization induction, under long-day conditions, vegetative growth shifts to reproductive growth, causing the fleshy root to stagnate, resulting in pithiness and fibrosis, severely reducing yield and edible value.

[0003] Currently, there is a shortage of high-quality bolting-resistant radish varieties in China, and the market is highly dependent on imported varieties from South Korea and Japan. Although imported varieties have good bolting resistance, their seeds are expensive, their adaptability is limited, their disease resistance is poor, and their supply chains are easily affected by fluctuations in the international market, significantly increasing the cost and risk of spring radish planting in my country. Therefore, the independent breeding and import substitution of high-quality, late-bolting, and disease-resistant new radish varieties has become one of the key focuses of current radish breeding work in China, and is of great practical significance for ensuring stable spring radish production, reducing planting costs, and enhancing the core competitiveness of the industry.

[0004] Radishes are typical cross-pollinated crops, and traditional breeding methods suffer from problems such as long breeding cycles, cumbersome and labor-intensive processes, and unstable genetic traits. Double haploid (DH) breeding technology, by inducing microspore embryogenesis, can obtain genotype-completely homozygous regenerated plants within a single generation, which can be directly used as parents, significantly shortening the breeding cycle and improving breeding efficiency. However, radish free microspore culture technology still faces technical bottlenecks such as strong genotype dependence, low microspore-derived embryogenesis rate, and difficulty in regeneration. This is especially true for late-bolting radish germplasm, which is a typical "genetically recalcitrant" material, making it almost impossible to obtain effective microspore-derived embryos under conventional microspore culture systems. This severely restricts the application of DH technology in the creation of superior germplasm and variety breeding of late-bolting radishes. Summary of the Invention

[0005] This invention aims to overcome the technical bottleneck of extremely low microspore-derived embryo formation rate and difficulty in obtaining effective microspore-derived embryos in the existing technology of microspore culture of late-bolting radish, and provides a method for rapidly creating new germplasm of late-bolting radish resistant to downy mildew. This method, through the synergistic effect of directional selection of parental materials, optimization of low-temperature pretreatment time, and exogenous NaB addition, can significantly improve the microspore-derived embryo formation rate of F1 material of late-bolting radish resistant to downy mildew, successfully creating a superior DH germplasm with completely homozygous genotype and possessing both late bolting and downy mildew resistance traits.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for rapidly creating new late-bolting, downy mildew-resistant radish germplasm includes the following steps:

[0008] (1) Hybridization to create F1 generation: Using downy mildew resistant radish material as the female parent and bolting resistant radish material as the male parent, hybridization was carried out to obtain F1 generation hybrid seeds;

[0009] (2) Vernalization treatment and screening: F1 seeds were germinated and then subjected to low-temperature vernalization treatment. After transplanting, F1 plants with late bolting phenotype and no downy mildew symptoms were selected as donor materials for microspore culture.

[0010] (3) Low temperature pretreatment of flower buds: flower buds of F1 plants obtained in step (2) at the edge stage of mononuclear stage were selected and subjected to low temperature pretreatment at 4℃.

[0011] (4) Culture of free microspores: The flower buds after pretreatment in step (3) are isolated and purified into microspores. The microspores are suspended in 1 / 2NLN-13 medium with sodium butyrate (NaB) added and cultured to induce microspore embryogenesis and obtain microspore-derived embryos.

[0012] (5) Microspore-derived embryo differentiation and plant regeneration: The microspore-derived embryos obtained in step (4) were successively subjected to differentiation culture, bud strengthening culture and rooting culture to obtain regenerated plants;

[0013] (6) Vernalization and field planting: The regenerated plants obtained in step (5) are hardened off and subjected to low-temperature vernalization before being planted in the field;

[0014] (7) Homozygosity analysis and target germplasm screening: InDel molecular markers combined with whole genome resequencing were used to perform whole genome homozygosity analysis on regenerated plants, and combined with field agronomic trait identification, DH single plants that were naturally doubled, genotype 100% homozygous and had both late bolting and downy mildew resistance were screened.

[0015] (8) Self-pollination and seed saving: Self-pollination and seed saving of the DH single plants obtained in step (7) to obtain DH germplasm of late bolting resistant radish with a completely homozygous genome.

[0016] Preferably, the downy mildew-resistant radish material in step (1) is the high-generation inbred line DM2, and the bolting-resistant radish material is the high-generation inbred line LB.

[0017] Preferably, the conditions for the low-temperature vernalization treatment are: treatment at 4°C for 35 days.

[0018] Preferably, the low-temperature pretreatment time is 24 hours.

[0019] Preferably, the concentration of NaB in step (4) in 1 / 2NLN-13 medium is 1.0 μM.

[0020] Preferably, the conditions for microspore culture in step (4) are: first heat shock treatment at 32.5℃ for 24~36 h, and then dark culture at 25℃ for 18~25 d.

[0021] Preferably, the conditions for the low-temperature vernalization treatment in step (6) are: temperature 8°C, alternating between light culture for 14 h and dark culture for 10 h, for a total of 25 days.

[0022] Preferably, the standard for homozygosity analysis in step (7) is: no heterozygous variation at any locus in the whole genome and genotype homozygosity of 100%.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. Overcoming the genotypic limitations of late-bolting radish: Addressing the technical bottleneck of inducing microspore embryogenesis in late-bolting radish germplasm using conventional methods, this invention improves the microspore-derived embryogenesis rate of this type of "genetically recalcitrant" material through the synergistic effect of parental selection, optimization of low-temperature pretreatment time, and exogenous NaB addition.

[0025] 2. Significantly shortened breeding cycle: Through DH technology, stable germplasm with 100% homozygous genome can be obtained within 1-2 generations, shortening the breeding cycle by more than 80% compared to conventional self-pollination purification methods (which usually require 6-8 generations).

[0026] 3. Precise aggregation of target traits: By using aggregation breeding methods and microspore culture technology, two important agronomic traits, late bolting and downy mildew resistance, are directionally aggregated into the same homozygous germplasm, achieving simultaneous improvement of multiple target traits and rapidly creating excellent DH homozygous germplasm with late bolting and downy mildew resistance.

[0027] 4. Improved breeding efficiency and accuracy: Homozygosity analysis using whole-genome resequencing combined with InDel molecular markers is more accurate and reliable than traditional morphological identification methods. Attached Figure Description

[0028] Figure 1 This is a breeding flowchart according to an embodiment of the present invention.

[0029] Figure 2 Microspore-derived embryos

[0030] Figure 3 Microspore-regenerated plants

[0031] Figure 4 Bolting-resistant DH plants

[0032] Figure 5 InDel markers are used to identify the homozygosity of DH plants.

[0033] Figure 6 Whole-genome resequencing to identify DH plant homozygosity Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The present invention will be further described in detail below with reference to the embodiments.

[0036] Donor parent material description

[0037] DM2, a high-generation inbred line of radish resistant to downy mildew, exhibits stable resistance to downy mildew.

[0038] LB, a high-generation self-pollinated line of bolting-resistant radish, exhibits stable late bolting characteristics.

[0039] Example 1

[0040] (1) Hybridization to create F1 generation: Using DM2, a high-generation inbred line of radish resistant to downy mildew, as the female parent and LB, a high-generation inbred line of high-quality late bolting radish, as the male parent, artificial hybridization was carried out during the budding stage to obtain F1 generation hybrid seeds.

[0041] (2) Vernalization treatment and screening: F1 seeds were vernalized in a 4℃ refrigerator for 35 days after germination and then sown in the field. F1 plants with late bolting phenotype and no downy mildew symptoms were selected as donor materials for microspore culture and used for subsequent free microspore culture.

[0042] (3) Low temperature pretreatment of flower buds: When the F1 plants reach the peak flowering period, take healthy and disease-free inflorescences, put them into a self-sealing bag, place moist absorbent cotton in the bag to maintain humidity, and pretreat them at 4℃ for 24 hours.

[0043] (4) Isolation and purification of microspores: After pretreatment, select uninucleate buds at the edge stage (2.5~3.5 mm), disinfect with 70% anhydrous ethanol for 30~45 s, disinfect with 5% sodium hypochlorite for 12~15 min, and rinse with sterile water 3~5 times. In B5 liquid extraction medium (B5 + 130 g / L sucrose + 15 g / L mannitol, pH 5.8), break the buds to release microspores, filter through a 40 μm sterile filter, centrifuge at 1200 rpm for 3 min, and discard the supernatant; resuspend the B5 extract and centrifuge again, repeat 3 times, and the resulting precipitate is the pure microspore.

[0044] (5) Culture of free microspores: The purified microspores were suspended in 1 / 2 NLN-13 medium supplemented with 1.0 μM NaB and heat-shocked at 32.5℃ for 24-36 h, and then cultured in the dark at 25℃ for 18-25 d. After the microspore-derived embryos were formed, they were transferred to light for culture.

[0045] (6) Embryoid germination and seedling formation: After the microspore-derived embryos turn green, they are transferred to B5 solid differentiation medium (B5 + 30 g / L sucrose + 8 g / L agar, pH 5.8~6.0). After 7 days, they are transferred to bud-strengthening medium (MS + 0.5 mg / L KT + 50 mL / L coconut juice + 30 g / L sucrose + 8 g / L agar, pH 5.8~6.0). When the seedlings are 3~5 cm tall, they are transferred to rooting medium (1 / 2MS + 0.5 mg / L IBA + 0.2 mg / L NAA + 30 g / L sucrose + 8 g / L agar, pH 5.8~6.0).

[0046] (7) Hardening off and transplanting and vernalization treatment: Hardening off seedlings was carried out after 25 days of rooting culture: sterile distilled water was added to the tissue culture bottle, the cap was opened halfway on the first day and fully opened on the second day. After 2-3 days, the plants were taken out, the agar was washed off, and the plants were transplanted into a nutrient soil:vermiculite = 1:2 (V / V) substrate for greenhouse cultivation. After the growth stabilized, the plants were transferred to an artificial climate chamber for vernalization: 8℃, alternating between 14 h of light culture and 10 h of dark culture, for 25 consecutive days. After the vernalization treatment, the plants were transferred to a greenhouse for 7 days of recovery, and then transplanted to the field.

[0047] (8) Homozygosity analysis and target germplasm screening: Morphological identification was performed on regenerated plants to confirm ploidy, and naturally doubled DH plants with normal plant type, normal floral organs, sufficient pollen, and normal self-pollination and seed setting were selected. Using donor F1 and haploids as controls, the homozygosity of regenerated plants was analyzed using InDel molecular markers and whole genome resequencing. Combined with field agronomic trait identification, naturally doubled doubled doubled DH plants with 100% homozygosity and both late bolting and downy mildew resistance were screened.

[0048] (9) Self-pollination and seed saving: The DH single plants selected above were self-pollinated and seed saving were obtained to obtain DH homozygous germplasm of radish that is resistant to downy mildew and late bolting.

[0049] Example 2: Effect of low-temperature pretreatment time on microspore-derived embryogenesis rate

[0050] This example was used to verify the effect of different low-temperature pretreatment times on the microspore-derived embryogenesis rate of F1 (DM2×LB). Except for the pretreatment time, all other operations were the same as in Example 1. Four time gradients were set for the low-temperature pretreatment: 0 h, 24 h, 36 h, and 48 h, with each treatment repeated three times. The results are as follows:

[0051] Table 1. Embryo emergence of F1 plants under different 4℃ low-temperature pretreatment times

[0052] 0 0.05 24 2.67 36 0.72 48 0.40

[0053] Results analysis: Without low-temperature pretreatment, the microspore-derived embryogenesis rate was extremely low (0.05 embryos / bud), making it almost impossible to obtain effective microspore-derived embryos. Pretreatment at 4℃ significantly increased the microspore-derived embryogenesis rate, showing an initial increase followed by a decrease, with the best effect observed after 24 h of low-temperature pretreatment (2.67 embryos / bud). These results indicate that 4℃ low-temperature pretreatment can effectively promote microspore embryogenesis in late-bolting radishes, and the optimal pretreatment time is 24 h.

[0054] Example 3: Effect of NaB concentration on embryo emergence rate

[0055] This example was used to verify the effect of NaB concentration on the microspore-derived embryogenesis rate of F1 (DM2×LB). The low-temperature pretreatment was fixed for 24 h, and all operations were the same as in Example 1 except for the NaB concentration. Three NaB concentration gradients were set: 0.5 μM, 1.0 μM, and 2.0 μM, with each treatment repeated three times. The results are as follows:

[0056] Table 2 Embryo emergence of F1 plants under different NaB concentrations

[0057] 0.5 0.20 1.0 2.93 2.0 0.91

[0058] Results analysis: Under the fixed 24 h low temperature pretreatment conditions, the microspore-derived embryogenesis rate was significantly increased after the addition of NaB, reaching a maximum of 2.93 embryos / buds at 1.0 μM, indicating that the addition of NaB and low temperature pretreatment have a significant synergistic effect, and the optimal addition concentration is 1.0 μM.

[0059] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for rapidly creating new germplasm of late bolting and downy mildew resistant radish, characterized by, Includes the following steps: (1) Hybridization to create F1 generation: Using downy mildew resistant radish material as the female parent and bolting resistant radish material as the male parent, hybridization was carried out to obtain F1 generation hybrid seeds; (2) Vernalization treatment and screening: F1 seeds were germinated and then subjected to low-temperature vernalization treatment. After transplanting, F1 plants with late bolting phenotype and no downy mildew symptoms were selected as donor materials for microspore culture. (3) Low temperature pretreatment of flower buds: flower buds of F1 plants obtained in step (2) at the edge stage of mononuclear stage were selected and subjected to low temperature pretreatment at 4℃. (4) Culture of free microspores: The flower buds after pretreatment in step (3) are isolated and purified into microspores. The microspores are suspended in 1 / 2NLN-13 medium with sodium butyrate (NaB) added and cultured to induce microspore embryogenesis and obtain microspore-derived embryos. (5) Microspore-derived embryo differentiation and plant regeneration: The microspore-derived embryos obtained in step (4) were successively subjected to differentiation culture, bud strengthening culture and rooting culture to obtain regenerated plants; (6) Vernalization and field planting: The regenerated plants obtained in step (5) are hardened off and subjected to low-temperature vernalization before being planted in the field; (7) Homozygosity analysis and target germplasm screening: The whole genome homozygosity of regenerated plants was analyzed by combining InDel molecular markers with whole genome resequencing, and combined with field agronomic trait identification, double haploid (DH) single plants that were naturally doubled, genotype 100% homozygous and had both late bolting and downy mildew resistance were screened. (8) Self-pollination and seed saving: The DH single plants obtained in step (7) are self-pollinated to obtain late bolting and downy mildew resistant radish DH homozygous germplasm.

2. The method for rapid creation of new germplasm of late bolting and downy mildew resistant radish according to claim 1, characterized in that: In step (1), the downy mildew resistant radish material is the high-generation inbred line DM2, and the bolting resistant radish material is the high-generation inbred line LB.

3. The method of quick creation of new germplasm of late bolting and downy mildew resistant radish according to claim 1, characterized in that: The conditions for the low-temperature vernalization treatment in step (2) are: treatment at 4℃ for 35 days.

4. The method for rapidly creating new late-bolting, downy mildew-resistant radish germplasm according to claim 1, characterized in that: The low-temperature pretreatment time in step (3) is 24 h.

5. The method for rapidly creating new late-bolting, downy mildew-resistant radish germplasm according to claim 1, characterized in that: The concentration of NaB in 1 / 2NLN-13 medium in step (4) is 1.0 μM.

6. The method for rapidly creating new late-bolting, downy mildew-resistant radish germplasm according to claim 1, characterized in that: The culture conditions described in step (4) are as follows: first, heat shock treatment at 32.5℃ for 24~36 h, and then dark culture at 25℃ for 18~25 d.

7. The method for rapidly creating new late-bolting, downy mildew-resistant radish germplasm according to claim 1, characterized in that: The conditions for the low-temperature vernalization treatment in step (6) are: temperature 8℃, alternating between light culture for 14 h and dark culture for 10 h, and continuous culture for 25 days.

8. The method for rapidly creating new late-bolting, downy mildew-resistant radish germplasm according to claim 1, characterized in that: The criteria for homozygosity analysis in step (7) are: no heterozygous variation at any locus in the whole genome and genotypic homozygosity of 100%.

9. The application of the method according to any one of claims 1-8 in accelerating the breeding of radish germplasm resistant to late bolting and downy mildew.

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