Method for creating new bolting-resistant radish germplasm by using microspore culture technology

Abstract

The invention discloses a method for creating a new bolting-resistant radish germplasm by using a microspore culture technology, and belongs to the technical field of radish breeding. The method comprises the following steps: hybridizing a radish material R-16 and a bolting-resistant radish material LR-75, carrying out microspore culture, embryoid induction and regeneration on hybrid offspring plants to obtain double haplobionts, carrying out selfing, and carrying out character evaluation and screening on selfing offspring plants to obtain bolting-resistant double haplobionts with stable characters. Experimental results show that by selecting the parent material and optimizing the hormone concentration of the induction culture medium, the embryo emergence rate of the radish material is remarkably increased, and the homozygous bolting-resistant radish germplasm can be efficiently and quickly obtained. According to the method, a rich material foundation is laid for heterosis breeding of the bolting-resistant radishes, the breeding procedure is simplified, the breeding cost is reduced, the breeding process of new varieties of the bolting-resistant radishes is accelerated, and the method has great economic significance and wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of radish breeding, in particular to a method for creating new bolting-resistant radish germplasm by utilizing microspore culture technology. Background Art

[0002] Currently, agricultural production relies primarily on imported bolting-resistant radish varieties. Domestically bred varieties lag behind imported varieties in bolting resistance and other quality traits. Therefore, breeding high-quality, bolting-resistant radish varieties to overcome the monopoly of imported bolting-resistant radish seed varieties has become an urgent challenge. Using free microspore culture technology to purify hybrid radish material, large numbers of genotypically rich, genetically stable doubled haploid plants can be obtained in just one to two years, effectively shortening the breeding cycle and improving breeding efficiency.

[0003] Microspore culture technology has been widely used in the breeding of cruciferous crops such as rapeseed and cabbage, but the current microspore culture system for radish is still imperfect. Issues such as strong genotype dependence, low embryoid induction rates, and difficulty in regenerating adventitious buds have limited its application in radish breeding. Bolting-resistant radishes are genetically recalcitrant, making embryos difficult to produce using conventional radish microspore culture systems.

[0004] Currently, there is a shortage of bolting-resistant radish germplasm resources. Existing studies have identified the bolting traits of 105 radish germplasms, of which only 22 are bolting-resistant, and most of them are imported varieties. Although various breeding institutions have developed a number of late-bolting radish varieties with excellent performance in recent years, the varieties bred through independent innovation still lag behind imported varieties in bolting resistance and other quality traits. The traditional breeding method often requires 6-8 generations of continuous self-pollination to select inbred lines with high combining ability and stability as parents for pairing. This traditional breeding method is not only time-consuming and labor-intensive, but continuous self-pollination will cause the materials to show obvious self-pollination depression. The use of free microspore culture technology to purify radish hybrid materials only takes 1-2 years to obtain a large number of double haploid plants with rich genotypes and genetic stability, which can effectively shorten the breeding period and improve breeding efficiency. However, the current microspore culture system for radish is still imperfect. Problems such as strong genotype dependence, low embryoid induction rates, and difficulty in regenerating adventitious buds limit its application in radish breeding. Furthermore, bolting-resistant radishes are genetically stubborn, making it difficult to produce embryos using conventional radish microspore culture systems. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for creating a new bolting-resistant radish germplasm using microspore culture technology to solve the problems existing in the above-mentioned prior art. The present invention significantly improves the embryo emergence rate of the radish material by selecting parent radish materials and optimizing the hormone concentration of the induction culture medium, laying a rich material foundation for the hybrid vigor breeding of bolting-resistant radish.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for creating bolting-resistant radish germplasm, comprising the following steps:

[0008] Crossing radish material R-16 with a bolting-resistant radish material to obtain F1 generation seeds, sowing them, screening for bolting-resistant traits, and obtaining F1 generation plants;

[0009] Taking the inflorescence of the F1 generation plant, selecting the donor flower buds by microscopic examination, disinfecting, and releasing microspores;

[0010] Purifying the microspores, resuspending in culture medium, heat shock treating, and inducing culture until embryoids appear;

[0011] Cultivating the embryoids until regenerated seedlings are obtained, hardening the regenerated seedlings, slowing down the seedling growth, and planting them in the field to obtain M0 generation plants;

[0012] The M0 generation plants are subjected to ploidy testing, diploid plants are selected, self-pollinated, M1 generation seeds are obtained, sown, M1 generation plants are obtained, and traits are comprehensively screened to obtain bolting-resistant radish plants, which are the bolting-resistant radish germplasm.

[0013] Preferably, the bolting-resistant radish material is radish material LR-75.

[0014] Preferably, the screening for the bolting resistance trait is to screen radish plants whose bolting time is no earlier than 160 days.

[0015] Preferably, the donor flower buds selected by microscopic examination are flower buds at the uninucleate marginal stage during microspore development.

[0016] Preferably, the culture medium is NLN-13 culture medium containing plant hormones and colchicine.

[0017] Preferably, the concentration of colchicine is 0.08 g / L;

[0018] The plant hormones are 6-benzylaminopurine and naphthaleneacetic acid;

[0019] The concentration of the 6-benzylaminopurine is 0.10 mg / L, and the concentration of the naphthaleneacetic acid is 1.5 mg / L.

[0020] Preferably, the temperature of the heat shock treatment is 32° C. and the time is 48 hours.

[0021] Preferably, the induction culture is a dark shaking culture at a temperature of 25° C. and a shaking speed of 40 rpm.

[0022] Preferably, the comprehensive screening of traits includes screening root traits and screening bolting resistance traits;

[0023] The root traits include: root shape, root diameter, root length and single root weight;

[0024] The screening of the bolting resistance trait is to screen radish plants whose bolting time is no earlier than 160 days.

[0025] The present invention also provides a use of the bolting-resistant radish plant obtained according to the above method in cultivating bolting-resistant radish varieties.

[0026] The present invention discloses the following technical effects:

[0027] The present invention hybridizes radish material R-16 with bolting-resistant radish material LR-75, subjects the hybrid progeny to microspore culture, embryoid induction, and regeneration to obtain double haploid plants, self-pollinates, and evaluates and screens the traits of the selfed progeny plants to obtain bolting-resistant double haploid plants with stable traits. Experimental results show that by selecting parental materials and optimizing the hormone concentration of the induction medium, the present invention reduces the differences in embryoid induction ability between genotypes, weakens the genotype restrictions imposed by bolting-resistant radish during microspore culture, and thus significantly improves the embryo emergence rate of the radish material, making it possible to efficiently and quickly obtain homozygous bolting-resistant radish germplasm. The present invention lays a rich material foundation for hybrid vigor breeding of bolting-resistant radish, simplifies breeding procedures, reduces breeding costs, and accelerates the selection process of new bolting-resistant radish varieties, thus having great economic significance and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1This figure illustrates the process of creating new bolting-resistant radish germplasm using microspore culture technology. A represents microspores extracted from anthers, with a scale of 20 μm. B represents enlarged microspores after 7-10 days of culture, with a scale of 20 μm. C represents cotyledonary embryoids after 4-6 weeks of culture, with a scale of 100 μm. D represents embryoids after 6-8 weeks of culture. E represents embryoids transferred to regeneration medium. F represents seedlings regenerated from embryoids. G represents rooted seedlings. H represents seeds from self-pollinated M0 plants. I represents M1 plants at different bolting stages.

[0030] Figure 2 The results of chromosome ploidy identification of regenerated plants are shown in Figure 1. A is the peak diagram for identification of parent LR-75; B is the peak diagram for identification of double haploid. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The present invention uses the F1 generation population of radish material R-16 and late-bolting material LR-75 as materials, narrows the differences in embryoid induction ability between genotypes, and weakens the genotype restrictions on bolting-resistant radish during microspore culture. Furthermore, by optimizing the microspore culture embryoid induction system and combining comprehensive evaluation of root traits and bolting-resistant traits, bolting-resistant double haploid pure line plants with excellent comprehensive traits are screened. The method of the present invention, to a certain extent, breaks the genotype restrictions on microspore embryoid induction, significantly improves the embryo emergence rate of the material, and quickly obtains homozygous bolting-resistant radish germplasm, which not only lays a rich material foundation for bolting-resistant radish heterosis breeding, but also effectively simplifies the breeding procedure, reduces breeding costs, and accelerates the selection process of new bolting-resistant radish varieties.

[0037] The radish material R-16 of the present invention is an inbred line of the collected and preserved local variety "Rugao Radish" that has been self-pollinated and purified for 4 generations. The radish material LR-75 of the present invention is the variety "Bai Ruyu" purchased from "Wuwei Jinsi Road Seed Industry Co., Ltd.". The radish material "Bai Yuchun" of the present invention is a widely planted commercial variety and has been disclosed in "Shi Zhengkan, Zhou Meixian, Bao Chonglai. Comparative test of introduction of 10 bolting-resistant radishes in Longquan [J]. Zhejiang Agricultural Science, 2023, 64(05): 1207-1209.". The applicant promises to distribute the above-mentioned biological material to the public within 3 years from the date of application of this invention.

[0038] The preparation methods of the NLN-13 culture medium and the B5 culture medium of the present invention have been disclosed in the document "Cong Jialin. Microspore culture of Mizuna and differential expression analysis of anthocyanin synthesis-related genes [D]. Shenyang Agricultural University, 2020.".

[0039] Example 1

[0040] This example optimizes the concentrations of hormones 6-benzylaminopurine (6-BA) and naphthaleneacetic acid (NAA) in NLN-13 culture medium to increase the embryoid induction rate. The setting scheme of 6-BA and NAA concentrations in NLN-13 culture medium is shown in Table 1.

[0041] R-16 was hybridized with LR-75, a low-generation inbred line of radish with high quality and bolting resistance, to obtain F1 generation seeds.

[0042] The F1 generation seeds were sown in the field. In March of the following year, individual plants of the F1 population, R-16 material and LR-75 material were selected, sampled and cultured for microspores.

[0043] The sampling and microspore culture methods are as follows:

[0044] When the radish plants grow to the early flowering stage (more than 5% of the flowers are open), remove the inflorescences on the main branches and primary branches for later use. The inflorescences can be stored at 4°C for 3-4 days.

[0045] Use tweezers to peel off the flower buds, pick the anthers and place them on a glass slide. Cover with a coverslip and squeeze to release the microspores. Observe under a microscope and select flower buds with most microspores in the uninucleate marginal stage (triangular shape) with a bud length of 2.5mm-3.0mm.

[0046] Take 50 buds from each tube, disinfect them with 75% alcohol for 30 seconds, 0.1% HgCl2 for 8 minutes, and rinse with sterile water three times.

[0047] The flower buds were ground in B5 medium (pH = 5.8) to release microspores, and the mixture was filtered through a 400-mesh stainless steel filter. The filtrate was collected in a 15 mL conical-bottom glass centrifuge tube, made up to 10 mL with B5 medium (pH = 5.8), and centrifuged at 800 rpm for 5 min. The supernatant was discarded.

[0048] The microspore pellet was resuspended in 8 mL of B5 medium (pH = 5.8), centrifuged at 800 rpm for 5 min, and the supernatant was discarded.

[0049] The microspore pellet was resuspended in 5 mL of NLN-13 medium (pH=5.8), centrifuged at 800 rpm for 5 min, and the supernatant was discarded.

[0050] The microspores were resuspended in 30 mL of NLN-13 medium (pH=5.8, 0.08 g / L colchicine) and evenly distributed into 18 culture dishes. The microspores were heat-shocked at 32° C. for 48 h and observed under a microscope.

[0051] The microspores were transferred to 25°C, 40 rpm, and cultured in the dark. The number of embryoids produced was counted and the embryo rate (embryoid induction rate) was calculated.

[0052] The results of embryo emergence rate are shown in Table 1. The embryo emergence rate of F1 plants was significantly higher than that of the bolting-resistant parent LR-75. Moreover, the embryo emergence rate of F1 generation was the highest under the plant hormone concentration of treatment 6 (6-BA 0.10 mg / L + NAA 1.5 mg / L). Therefore, subsequent experiments used NLN-13 medium with a hormone formula of 6-BA 0.10 mg / L + NAA 1.5 mg / L for embryoid induction and other cultures.

[0053] Table 1 Plant hormone concentration screening results

[0054]

[0055] Example 2

[0056] The NLN-13 culture medium of this example contains 0.10 mg / L 6-BA and 1.5 mg / L NAA.

[0057] 1. Hybridization of radish materials

[0058] R-16 was hybridized with LR-75, a low-generation inbred line of radish with high quality and bolting resistance, to obtain F1 generation seeds.

[0059] The F1 generation seeds were sown in the field in early September of the first year, and the bolting characteristics of the F1 population were investigated in March of the second year. 35 bolting-resistant individual plants were selected for mixed sampling and microspore culture.

[0060] The bolting time of Baiyuchun is 165-170 days. Materials whose bolting time is no more than 5 days earlier than that of Baiyuchun or later than that of Baiyuchun are judged as bolting-resistant materials.

[0061] 2. Sampling and Microspore Culture Methods

[0062] After the bolting-resistant radish plants in the F1 population grow to the early flowering stage (more than 5% of the flowers are open), the inflorescences on the main branches and primary branches are taken for later use. The inflorescences can be stored at 4°C for 3-4 days.

[0063] Use tweezers to peel off the flower buds, pick the anthers and place them on a glass slide. Cover with a coverslip and squeeze to release the microspores. Observe under a microscope and select flower buds with most microspores in the uninucleate marginal stage (triangular shape) with a bud length of 2.5mm-3.0mm.

[0064] Take 50 buds from each tube, disinfect them with 75% alcohol for 30 seconds, 0.1% HgCl2 for 8 minutes, and rinse with sterile water three times.

[0065] The flower buds were ground in B5 medium (pH = 5.8) to release microspores, and the mixture was filtered through a 400-mesh stainless steel filter. The filtrate was collected in a 15 mL conical-bottom glass centrifuge tube, made up to 10 mL with B5 medium (pH = 5.8), and centrifuged at 800 rpm for 5 min. The supernatant was discarded.

[0066] The microspore pellet was resuspended in 8 mL of B5 medium (pH = 5.8), centrifuged at 800 rpm for 5 min, and the supernatant was discarded.

[0067] The microspore pellet was resuspended in 5 mL of NLN-13 medium (pH=5.8), centrifuged at 800 rpm for 5 min, and the supernatant was discarded.

[0068] The microspores were resuspended in 30 mL NLN-13 medium (pH = 5.8, 0.08 g / L colchicine) and evenly distributed into 18 culture dishes. They were heat-shocked at 32 ° C for 48 h and observed under a microscope. The results were as follows: Figure 1 As shown in A.

[0069] The microspores were transferred to 25°C, 40 rpm, and cultured in the dark until embryoids appeared. The culture process was as follows: Figure 1 As shown in B, C, and D.

[0070] The embryoids were cultured under light conditions (25°C, 16 h light, 8 h dark) until they turned green.

[0071] 3. Cultivation Method of Regenerated Seedlings

[0072] The green embryoids were transferred to B5 solid medium (pH = 5.8) and cultured at 25 ° C, 16 h light, 8 h dark to produce regenerated seedlings. The medium was changed every two weeks. The culture results were as follows: Figure 1 As shown in E.

[0073] After the embryoids regenerate into regenerated seedlings, the regenerated seedlings are transferred to MS solid medium (pH = 5.8) for subculture. Figure 1 As shown in F.

[0074] When the regenerated seedlings grew to about 3-5 cm, they were transferred to MS rooting medium (pH = 5.8). Figure 1 As shown in G.

[0075] When the root system of the regenerated seedlings is well developed, the regenerated seedlings are hardened. After hardening, the regenerated seedlings are transplanted into nutrient soil, bagged to maintain humidity, and allowed to grow slowly. After successful growth, the bags can be gradually removed.

[0076] 4. Cultivation Methods of M0 and M1 Generation Plants

[0077] After about one month of acclimatization, the regenerated seedlings were transplanted to the experimental field for daily management, which were the double haploid M0 generation plants. The leaves of the M0 generation plants were taken for chromosome ploidy test, and the results were as follows: Figure 2 As shown; the regenerated seedlings (M0) with a diploid chromosome ploidy were self-pollinated, as shown Figure 1 As shown in middle H, M1 generation seeds were obtained.

[0078] The M1 generation seeds were sown in the experimental field in the autumn and winter of the second year. The Korean excellent bolting-resistant commercial radish variety 'Baekyochun' was used as a control. The root system comprehensive traits (root shape, root diameter, root length, single root weight) and bolting time (the number of days from the date of sowing to the plant bolt height of 3 cm) of the double haploid M1 generation plants were investigated and evaluated. Figure 1 As shown in Figure 1. The bolting time of Baiyuchun is 165-170 days. Materials with bolting time no more than 5 days earlier than Baiyuchun or later than Baiyuchun are judged as bolting-resistant materials.

[0079] The results are shown in Table 2. Among the M1 generation radish plants, M1-4, M1-5, M1-6, M1-7, M1-8, M1-9, M1-12 and M1-13 are all bolting-resistant materials, and the ploidy is diploid. Among the 13 materials, 8 bolting-resistant materials were obtained, indicating that the method provided by the present invention can efficiently create bolting-resistant radish materials.

[0080] Table 2 Agronomic traits, bolting resistance and ploidy test results of M1 generation radish

[0081] Material number Root type Root length / cm Root diameter / cm Single weight / g Bolting time / d ploidy <![CDATA[M1-1]]> Long cylinder 19.6 4.34 270 141 Diploid <![CDATA[M1-2]]> Long cylinder 16.1 5.46 363 153 Diploid <![CDATA[M1-3]]> Short cylinder 13.5 4.83 215 159 Diploid <![CDATA[M1-4]]> Long cylinder 19.3 5.91 513 174 Diploid <![CDATA[M1-5]]> Long cylinder 15.3 4.89 286 167 Diploid <![CDATA[M1-6]]> spindle-shaped 15.7 5.34 237 160 Diploid <![CDATA[M1-7]]> Short cylinder 12.7 4.82 231 182 Diploid <![CDATA[M1-8]]> Short cylinder 13.6 4.61 228 174 Diploid <![CDATA[M1-9]]> Long cylinder 14.8 3.72 171 176 Diploid <![CDATA[M1-10]]> spindle-shaped 16.3 3.97 134 155 Diploid <![CDATA[M1-11]]> spindle-shaped 10.2 4.43 106 153 Diploid <![CDATA[M1-12]]> spindle-shaped 11.2 5.66 182 171 Diploid <![CDATA[M1-13]]> Short cylinder 11.7 3.15 91 174 Diploid

[0082] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for creating bolting-resistant radish germplasm, characterized in that: The following steps are involved: Crossing radish material R-16 with a bolting-resistant radish material to obtain F1 generation seeds, sowing them, screening for bolting-resistant traits, and obtaining F1 generation plants; Taking the inflorescence of the F1 generation plant, selecting the donor flower buds by microscopic examination, disinfecting, and releasing microspores; Purifying the microspores, resuspending in culture medium, heat shock treating, and inducing culture until embryoids appear; Cultivating the embryoids until regenerated seedlings are obtained, hardening the regenerated seedlings, slowing down the seedling growth, and planting them in the field to obtain M0 generation plants; The M0 generation plants are subjected to ploidy testing, diploid plants are selected, self-pollinated, M1 generation seeds are obtained, sown, M1 generation plants are obtained, and traits are comprehensively screened to obtain bolting-resistant radish plants, which are the bolting-resistant radish germplasm.

2. The method according to claim 1, wherein The bolting-resistant radish material is radish material LR-75.

3. The method according to claim 1, wherein The screening of the bolting resistance trait is to screen radish plants whose bolting time is no earlier than 160 days.

4. The method according to claim 1, wherein The donor flower buds selected for microscopic examination are flower buds at the uninucleate marginal stage during the microspore development period.

5. The method according to claim 1, wherein The culture medium is NLN-13 culture medium containing plant hormones and colchicine.

6. The method according to claim 5, wherein The concentration of colchicine is 0.08g / L; The plant hormones are 6-benzylaminopurine and naphthaleneacetic acid; The concentration of the 6-benzylaminopurine is 0.10 mg / L, and the concentration of the naphthaleneacetic acid is 1.5 mg / L.

7. The method according to claim 1, wherein The temperature of the heat shock treatment is 32° C. and the time is 48 h.

8. The method according to claim 1, wherein The induction culture was performed in a dark shaking culture at a temperature of 25° C. and a shaking speed of 40 rpm.

9. The method according to claim 1, wherein The comprehensive screening of traits includes screening root traits and screening bolting resistance traits; The root traits include: root shape, root diameter, root length and single root weight; The screening of the bolting resistance trait is to screen radish plants whose bolting time is no earlier than 160 days.

10. Use of the bolting-resistant radish plant obtained by the method according to any one of claims 1 to 9 in breeding bolting-resistant radish varieties.

Images