Method for directly obtaining haplobiont through tomato anther culture and culture medium

Abstract

The invention relates to a method and a culture medium for directly obtaining haplobionts through tomato anther culture, and belongs to the technical field of plant tissue culture. The method comprises the following steps: (1) obtaining an embryoid; and (2) obtaining the regenerated haplobiont. The culture medium used in the method is an anther culture solid culture medium and a healthy seedling rooting solid culture medium. The method has the advantages that the steps of the culture method are simplified, the operation is convenient, and the haplobiont is obtained by directly inducing the embryoid through anther in-vitro culture; as the method adopted by the invention does not need a callus stage, the tedious process of callus induction, transfer to a differential medium and induction of a regenerated plant is omitted; according to the method, ploidy mixing caused by the fact that diploid somatic cells such as anther walls and filaments participate in callus formation is completely eradicated from the development path, it is ensured that all obtained plants are haploids from pollen, and the purity and obtaining efficiency of haploid materials are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method and culture medium for directly obtaining haploid plants from tomato anther culture. Background Technology

[0002] Anther culture, as a primary method for obtaining haploid plants, holds significant value in plant breeding and genetic research. By inducing pollen cells to develop into haploid plants and then doubling chromosomes, homozygous diploid materials can be rapidly obtained. Compared to traditional hybridization breeding methods that require multiple generations of self-pollination and purification, this significantly shortens the breeding cycle (typically by 3-5 generations) and greatly improves the selection efficiency of target traits.

[0003] The exploration of tomato anther culture technology began in 1971, when Sharp et al. first induced haploid callus from immature tomato pollen. In 1972, Greshhoff and Doy obtained callus through anther culture and, after differentiation, first obtained haploid tomato plants. In recent years, there have been few research reports on obtaining haploid regenerated plants from tomato anther and free microspore culture, both domestically and internationally. Related studies mainly focus on: 1) the correspondence between pollen development stage and flower bud morphological characteristics; 2) the effect of flower bud stress treatment on callus induction; 3) the influence of genotype and culture medium composition on callus induction and differentiation; 4) the obvious ploidy mixing phenomenon in callus induced by anther culture; and 5) exploring the possible origin of regenerated plants by comparing the morphological characteristics and molecular markers of regenerated plants and donor plants. Currently, tomato anther culture still faces significant bottlenecks: strong genotype dependence, with most varieties showing weak responses to culture conditions; low callus induction and differentiation rates with mixed ploidy; and embryoids arresting in early development during free microspore culture, making it difficult to obtain complete plants. These problems severely restrict the large-scale application of tomato haploid breeding technology, necessitating systematic research on genotype screening, culture system optimization, and molecular mechanism analysis.

[0004] Significant limitations of relevant domestic and international research include: some reports show that regenerated plants have not undergone systematic testing, making it impossible to determine their ploidy and limiting the reliability of the results; all successful cases of tomato anther culture obtained regenerated plants through the callus route, which requires two stages: "dedifferentiation to form callus tissue → callus tissue differentiation into embryoids / adventitious buds," and requires transfer to two different culture media, resulting in a large workload and complicated procedures, and there are no reports of anther culture producing seedlings in one step. Summary of the Invention

[0005] The first objective of this invention is to disclose a method for directly obtaining haploid plants from tomato anther culture.

[0006] The second objective of this invention is to disclose the solid culture medium for anther culture used in the above-described method.

[0007] The third objective of this invention is to disclose the solid culture medium for healthy seedling rooting used in the above method.

[0008] The objective of this invention is achieved through the following technical solution: A method for directly obtaining haploid plants from tomato anther culture, comprising the following steps: (1) Obtaining embryoids: Select sterile anthers in the uninucleate or binucleate stage, inoculate them into anther culture solid medium, and culture them in the dark at 35℃ for 4 days. Then, culture them in the light at 25-28℃ for 50-90 days. The embryoids can be seen to germinate from the split anther wall. The composition of the solid culture medium for anther culture is as follows: 1900 mg / L KNO3, 1650 mg / L NH4NO3, 332.02 mg / L CaCl2, 180.54 mg / L MgSO4, 170 mg / L KH2PO4, 37.3 mg / L Na2·EDTA, 27.8 mg / L Fe2SO4·7H2O, 6.2 mg / L H3BO3, 16.9 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 100 mg / L inositol, 1.0 mg / L nicotinic acid, 1.0 mg / L glycine, and 10 mg / L thiamine hydrochloride (vitamin B1). 0.1–1 mg / L KT, 0.05–2 mg / L ABA, 3–6% sucrose and 0.6–0.8% agar powder; the pH of the solid medium for anther culture is 5.8–6.2; (2) Obtaining regenerated haploid plants: When the embryoid grows to the cotyledon embryoid stage, it is transferred to a healthy seedling rooting solid culture medium for continued culture until it grows into a complete plant. The composition of the solid culture medium for healthy seedling rooting is as follows: 1900 mg / L KNO3, 1650 mg / L NH4NO3, 332.02 mg / L CaCl2, 180.54 mg / L MgSO4, 170 mg / L KH2PO4, 37.3 mg / L Na2·EDTA, 27.8 mg / L Fe2SO4·7H2O, 0.83 mg / L KI, 6.2 mg / L H3BO3, 16.9 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 100 mg / L inositol, 0.5 mg / L nicotinic acid, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride (vitamin B1), 0.5 mg / L pyridoxine hydrochloride (vitamin B6), 0.01–0.1 mg / L IAA, 0.1–0.5 mg / L NAA, 2% sucrose, 0.8% agar; the pH of the solid medium for healthy seedling rooting is 5.8.

[0009] The above-described method for directly obtaining haploid plants from tomato anther culture includes the following steps prior to the above method: (1) Selection of fresh flower buds: Select flower buds in the uninucleate marginal stage or binucleate stage of microspores; (2) Pretreatment of flower buds: Treat at 4℃ for 2 days; (3) Surface disinfection of flower buds: Soak flower buds in 75% alcohol for 0.5 to 1 minute, soak them in 5% sodium hypochlorite solution for 3 to 5 minutes, rinse them 3 times with sterile water, and dry them with sterile filter paper.

[0010] The anther culture solid culture medium used in the above-described technical solution comprises the following components: 1900 mg / L KNO3, 1650 mg / L NH4NO3, 332.02 mg / L CaCl2, 180.54 mg / L MgSO4, 170 mg / L KH2PO4, 37.3 mg / L Na2·EDTA, 27.8 mg / L Fe2SO4·7H2O, 6.2 mg / L H3BO3, 16.9 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, and 0.025 mg / L Na2MoO4·2H2O. The ingredients are: CoCl2·6H2O, 100 mg / L inositol, 1.0 mg / L nicotinic acid, 1.0 mg / L glycine, 10 mg / L thiamine hydrochloride (vitamin B1), 0.1–1 mg / L KT, 0.05–2 mg / L ABA, 3–6% sucrose, and 0.6–0.8% agar powder; the pH of the solid medium for anther culture is 5.8–6.2.

[0011] The culture medium for healthy seedling rooting solid used in the cultivation method described in the above technical solution comprises: 1900 mg / L KNO3, 1650 mg / L NH4NO3, 332.02 mg / L CaCl2, 180.54 mg / L MgSO4, 170 mg / L KH2PO4, 37.3 mg / L Na2·EDTA, 27.8 mg / L Fe2SO4·7H2O, 0.83 mg / L KI, 6.2 mg / L H3BO3, 16.9 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, and 0.025 mg / L... CoCl2·6H2O, 100 mg / L inositol, 0.5 mg / L nicotinic acid, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride (vitamin B1), 0.5 mg / L pyridoxine hydrochloride (vitamin B6), 0.01–0.1 mg / L IAA, 0.1–0.5 mg / L NAA, 2% sucrose, 0.8% agar; the pH of the solid medium for healthy seedling rooting is 5.8.

[0012] The present invention has the following beneficial effects: 1. The culture method of this invention is simplified and convenient to operate, directly inducing embryoids through anther culture to obtain haploid plants. Since the method of this invention does not require a callus stage, it eliminates the cumbersome process of "callus induction → transfer to differentiation medium → induction of regenerated plants".

[0013] 2. This invention eliminates the ploidy mixture caused by diploid somatic cells such as anther walls and filaments participating in callus formation from the developmental path, ensuring that the obtained plants are all haploids derived from pollen, and significantly improving the purity and acquisition efficiency of haploid materials. Attached Figure Description

[0014] Figure 1 Anther culture of the large-fruited tomato variety "Hard Powder 8" as described in Example 1. Wherein: A represents embryoids directly obtained from anther culture induction; B represents embryoid development; C represents the formation of regenerated plantlets; and D represents the rooting and vigorous growth of the regenerated plantlets.

[0015] Figure 2 Anther culture of the cherry tomato variety Jingdan 5 as described in Example 2. Wherein: A represents embryoids directly obtained from anther culture induction; B represents rooted and robust seedlings of regenerated plantlets. Detailed Implementation

[0016] To facilitate understanding of the technical solution of the present invention, the following detailed description, in conjunction with specific embodiments, further illustrates the method and culture medium for directly obtaining haploid plants from tomato anther culture according to the present invention.

[0017] Unless otherwise specified, all experiments in the following examples were conducted using conventional methods in the field. The experimental materials, large-fruited tomato No. 8 and cherry tomato No. 5, were grown in the plastic greenhouse of Shangzhuang Agricultural Science and Technology Park, Plant Tissue Culture Technology Laboratory, Haidian District, Beijing. Example 1

[0018] Anther culture of large-fruited tomato variety Hard Powder No. 8: From May to July 2023, between 7:00 AM and 8:00 AM, healthy flower buds free from pests and diseases were collected from robust plants and transported back to the laboratory in ice packs for 2 days at 4°C. Pollen was stained with DAPI (4,6-biamidin-2-phenylindole) to confirm the developmental stage as either uninucleate or binucleate. Flower buds were surface-sterilized with 75% alcohol for 1 minute, 5% sodium hypochlorite for 5 minutes, rinsed three times with sterile water, and blotted dry with sterile filter paper to obtain sterile flower buds. The anthers were removed from the flower buds using small forceps and placed on sterile anther culture medium. After 4 days of incubation in the dark at 35°C, the culture was transferred to 25°C under light for further incubation. After approximately 70 days, white embryoids were visible to the naked eye germinating from the split anther walls. When the embryoids differentiated into two small cotyledons (e.g., ...), ... Figure 1(As shown in A) it was transferred to a healthy seedling rooting solid culture medium for further cultivation (as shown in A) Figure 1 (as shown in B), cultured until... Figure 1 After the regenerated plantlets shown in C are formed, they are then strengthened and rooted (as shown in Figure C). Figure 1 (As shown in D), robust regenerated plants were finally obtained, which were identified as haploid plants by flow cytometry. Deformed plants could recover their normal phenotype after one subculture on this medium.

[0019] The solid culture medium for anther culture consisted of: 1900 mg / L KNO3, 1650 mg / L NH4NO3, 332.02 mg / L CaCl2, 180.54 mg / L MgSO4, 170 mg / L KH2PO4, 37.3 mg / L Na2·EDTA, 27.8 mg / L Fe2SO4·7H2O, 6.2 mg / L H3BO3, 16.9 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 100 mg / L inositol, 1.0 mg / L nicotinic acid, 1.0 mg / L glycine, 10 mg / L thiamine hydrochloride (vitamin B1), and 1 mg / L... KT, 0.1 mg / L ABA, 3% sucrose and 0.6% agar powder; the pH of the solid culture medium is 6.0; The solid culture medium for healthy seedling rooting consisted of: 1900 mg / L KNO3, 1650 mg / L NH4NO3, 332.02 mg / L CaCl2, 180.54 mg / L MgSO4, 170 mg / L KH2PO4, 37.3 mg / L Na2·EDTA, 27.8 mg / L Fe2SO4·7H2O, 0.83 mg / L KI, 6.2 mg / L H3BO3, 16.9 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, and 0.025 mg / L... CoCl2·6H2O, 100 mg / L inositol, 0.5 mg / L nicotinic acid, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride (vitamin B1), 0.5 mg / L pyridoxine hydrochloride (vitamin B6), 0.05 mg / L IAA, 0.1 mg / L NAA, 2% sucrose, 0.8% agar; the pH of the solid medium for healthy seedling rooting is 5.8. Example 2

[0020] Anther culture of cherry tomato Jingdan No. 5 Flower buds collected from a plastic greenhouse between 7:00 and 8:00 AM from May to July 2023 were transported back to the laboratory in ice packs and treated at 4°C for 2 days. Pollen was examined under a microscope to confirm the developmental stage as either uninucleate or binucleate. The flower buds were surface-sterilized with 75% alcohol for 1 minute, 5% sodium hypochlorite for 3 minutes, rinsed three times with sterile water, and dried with sterile filter paper to obtain sterile buds. The anthers were removed from the buds using small forceps and placed on anther culture medium. After 4 days of incubation in the dark at 35°C, the culture was transferred to 25°C under light for further incubation. Cotyledonous embryoids (such as...) were obtained after approximately 80 days. Figure 2 As shown in A), it was transferred to a healthy seedling rooting solid culture medium for continued culture, simultaneously completing the seedling strengthening and rooting process (as shown in A). Figure 2 (As shown in B), robust regenerated plants were finally obtained, which were identified as haploid plants by flow cytometry.

[0021] The composition of the sterile anther culture medium was as follows: 1900 mg / L KNO3, 1650 mg / L NH4NO3, 332.02 mg / L CaCl2, 180.54 mg / L MgSO4, 170 mg / L KH2PO4, 37.3 mg / L Na2·EDTA, 27.8 mg / L Fe2SO4·7H2O, 6.2 mg / L H3BO3, 16.9 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 100 mg / L inositol, 1.0 mg / L nicotinic acid, 1.0 mg / L glycine, and 10 mg / L thiamine hydrochloride (vitamin B1). 1 mg / L KT, 0.1 mg / L ABA, 3% sucrose and 0.6% agar powder; pH of the solid culture medium was 6.0; The solid culture medium for healthy seedling rooting consisted of: 1900 mg / L KNO3, 1650 mg / L NH4NO3, 332.02 mg / L CaCl2, 180.54 mg / L MgSO4, 170 mg / L KH2PO4, 37.3 mg / L Na2·EDTA, 27.8 mg / L Fe2SO4·7H2O, 0.83 mg / L KI, 6.2 mg / L H3BO3, 16.9 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, and 0.025 mg / L... CoCl2·6H2O, 100 mg / L inositol, 0.5 mg / L nicotinic acid, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride (vitamin B1), 0.5 mg / L pyridoxine hydrochloride (vitamin B6), 0.1 mg / L IAA, 0.1 mg / L NAA, 2% sucrose, 0.8% agar; the pH of the solid medium for healthy seedling rooting is 5.8.

[0022] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention in any form or substance. Any modifications, alterations, and variations made by those skilled in the art without departing from the scope of the present invention using the disclosed technical content are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and variations made to the above embodiments based on the essential technology of the present invention are still within the scope of the present invention.

Claims

1. A method for obtaining haploid plants directly from tomato anther culture, comprising the following steps: (1) Obtaining of embryoids: selecting sterile anthers at the single nuclear side or double nuclear stage, inoculating into anther culture solid medium, first culturing at 35℃ in dark for 4 days, then transferring to 25-28℃ under light for 50-90 days, and then embryoids can be observed to germinate from the split anther wall; The composition of the anther culture solid medium is: 1900 mg / L KNO3, 1650 mg / L NH4NO3, 332.02 mg / L CaCl2, 180.54 mg / L MgSO4, 170 mg / L KH2PO4, 37.3 mg / L Na2·EDTA, 27.8 mg / L Fe2SO4·7H2O, 6.2 mg / L H3BO3, 16.9 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 100 mg / L myo-inositol, 1.0 mg / L nicotinic acid, 1.0 mg / L glycine, 10 mg / L thiamine hydrochloride, 0.1-1 mg / L KT, 0.05-2 mg / L ABA, 3-6% sucrose and 0.6-0.8% agar powder; the pH value of the anther culture solid medium is 5.8-6.2; (2) Obtaining of regenerated haploid plants: when the embryoids grow to the cotyledon stage, transferring them to the healthy seedling rooting solid medium for further culturing, and then growing into complete plants; The composition of the healthy seedling rooting solid medium is: 1900 mg / L KNO3, 1650 mg / L NH4NO3, 332.02 mg / L CaCl2, 180.54 mg / L MgSO4, 170 mg / L KH2PO4, 37.3 mg / L Na2·EDTA, 27.8 mg / L Fe2SO4·7H2O, 0.83 mg / L KI, 6.2 mg / L H3BO3, 16.9 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 100 mg / L myo-inositol, 0.5 mg / L nicotinic acid, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, 0.01-0.1 mg / L IAA, 0.1-0.5 mg / L NAA, 2% sucrose, 0.8% agar; the pH value of the healthy seedling rooting solid medium is 5.

8.

2. The method for obtaining haploid plants directly from tomato anthers culture according to claim 1, characterized by the fact that, The method further comprises the following steps before the method: (1) Selecting of fresh flower buds: selecting flower buds with single nuclear side or double nuclear stage; (2) Pretreatment of flower buds: low temperature treatment at 4℃ for 2 days; (3) surface sterilization of the flower bud: the flower bud is soaked in 75% alcohol for 0.5-1 minute, 5% sodium hypochlorite solution for 3-5 minutes, and then washed with sterile water for 3 times, and dried with sterile filter paper.

3. Anther culture solid medium used in the culture method described in claim 1 or 2, characterized by: The composition of the anther culture solid medium is: 1900 mg / L KNO3, 1650 mg / L NH4NO3, 332.02 mg / L CaCl2, 180.54 mg / L MgSO4, 170 mg / L KH2PO4, 37.3 mg / L Na2·EDTA, 27.8 mg / L Fe2SO4·7H2O, 6.2 mg / L H3BO3, 16.9 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 100 mg / L myo-inositol, 1.0 mg / L nicotinic acid, 1.0 mg / L glycine, 10 mg / L thiamine hydrochloride, 0.1-1 mg / L KT, 0.05-2 mg / L ABA, 3-6% sucrose, and 0.6-0.8% agar powder; the pH value of the anther culture solid medium is 5.8-6.

2.

4. The solid medium for use in the culture method as claimed in claim 1 or 2, characterized in that: The composition of the seedling growth and rooting solid medium is: 1900 mg / L KNO3, 1650 mg / L NH4NO3, 332.02 mg / L CaCl2, 180.54 mg / L MgSO4, 170 mg / L KH2PO4, 37.3 mg / L Na2·EDTA, 27.8 mg / L Fe2SO4·7H2O, 0.83 mg / L KI, 6.2 mg / L H3BO3, 16.9 mg / L MnSO4·H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 100 mg / L myo-inositol, 0.5 mg / L nicotinic acid, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, 0.01-0.1 mg / L IAA, 0.1-0.5 mg / L NAA, 2% sucrose, and 0.8% agar; the pH value of the seedling growth and rooting solid medium is 5.8.

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