A haploid doubling composition and a haploid young embryo doubling method

Abstract

This invention provides a haploid doubling composition and a method for doubling haploid embryos, belonging to the field of crop breeding technology. By combining sorbitol, colchicine, and dimethyl sulfoxide (DMSO), this invention significantly improves the doubling rate when treating haploid plant materials, effectively solving the problem of low haploid embryo doubling efficiency in traditional colchicine treatment methods. The unique formulation provided by this invention can greatly increase the doubling pollen shedding rate and seed setting rate of haploid plants, thereby greatly improving the production efficiency of double haploids (DH). Through the application of this invention, an efficient and practical haploid breeding technology solution can be provided for the field of plant breeding, thereby significantly improving the overall efficiency of haploid breeding technology.

Description

Technical Field

[0001] This invention relates to the field of crop breeding technology, and in particular to a haploid doubling composition and a method for doubling haploid embryos. Background Technology

[0002] Haploid breeding technology comprises four parts: haploid induction, identification, doubling, and DH management. Haploid induction and identification are determined by haploid induction lines and corresponding haploid identification markers. With the cloning of haploid induction genes and the use of R1nj markers, the large-scale acquisition of haploids is no longer the rate-limiting step in haploid breeding. Haploid doubling efficiency is a crucial aspect of haploid breeding; that is, DH production efficiency is one of the important factors affecting haploid breeding efficiency.

[0003] Currently, the most widely used haploid doubling methods are bud soaking and haploid embryo tissue culture doubling. Compared to bud soaking, haploid embryo doubling is more efficient and is one of the best methods for industrialized DH production. Efficient doubling methods also need to be combined with efficient doubling reagents. Commonly used haploid doubling reagents include colchicine and herbicides. Colchicine has a significantly better doubling effect than herbicides and is widely used. Haploid doubling involves both male and female spike doubling. Male spike doubling directly determines the pollen shedding rate after treatment, while female spike doubling determines the seed setting rate. Haploids need both male and female spikes to double simultaneously to achieve normal seed setting and obtain DH. Therefore, ensuring high pollen shedding and seed setting rates in haploids is key to improving haploid doubling efficiency.

[0004] Chinese patent CN105918116A discloses a method for doubling plant haploids, which includes soaking the radicle of treated plant haploid buds in a mixture of colchicine (0.02%-0.05% by mass) and dimethyl sulfoxide (2%) for 18-36 hours at a temperature of 14-18°C.

[0005] However, the doubling effect of existing plant haploid doubling methods still needs to be improved. Summary of the Invention

[0006] The purpose of this invention is to provide a haploid doubling composition and a haploid embryo doubling method, which has higher doubling efficiency.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] The present invention provides a haploid doubling composition comprising colchicine, DMSO and sorbitol.

[0009] The present invention also provides the application of the above-mentioned haploid doubling composition in improving the haploid doubling efficiency of plant embryos or in plant haploid breeding.

[0010] Preferably, the plant is corn, rice, potato, sugar beet or wheat.

[0011] The present invention also provides a haploid doubling culture medium containing the above-mentioned haploid doubling composition.

[0012] Preferably, the final concentration of colchicine in the haploid doubling culture medium is 0.01–2 g / L;

[0013] In the haploid doubling culture medium, the final volume percentage concentration of DMSO is 1-5%.

[0014] The final concentration of sorbitol in the haploid doubling culture medium is 0.5–20 g / L.

[0015] Preferably, the haploid doubling culture medium further includes basal culture medium salt components, sucrose, agar, and water.

[0016] In this invention, the basal culture medium salt components refer to a series of inorganic salt compounds used to prepare the basal culture medium. These are the core components of the culture medium, providing the inorganic nutrients necessary for the growth of microorganisms or cells. These include macroelements such as nitrogen, phosphorus, potassium, sulfur, magnesium, and calcium, as well as microelements such as iron, zinc, manganese, and copper. They are usually provided in dry powder form and need to be accurately weighed according to a specific formula ratio and dissolved in water before use to prepare the required culture medium. The formulation ratios and types of these salt components will vary according to the specific needs of different culture subjects to meet their specific growth and metabolic conditions.

[0017] Preferably, the salt components of the basal culture medium are 1 / 2 MS medium basal salts or MS medium basal salts.

[0018] Preferably, in the haploid doubling culture medium, the final concentration of the salt components of the basal culture medium is 3-5 g / L;

[0019] In the haploid doubling culture medium, the final concentration of sucrose is 10–50 g / L;

[0020] In the haploid doubling medium, the final concentration of agar is 5–10 g / L.

[0021] Preferably, the pH of the haploid doubling culture medium is 5.5 to 7.

[0022] The present invention also provides a method for doubling haploid embryos, comprising the following steps:

[0023] The plant embryos were inoculated into the above-mentioned haploid doubling medium for induction culture to obtain haploid embryos;

[0024] The induction culture time is 10-30 hours.

[0025] The beneficial effects of this invention are:

[0026] This invention achieves a significant increase in doubling rate when treating haploid plant materials by mixing sorbitol, colchicine, and dimethyl sulfoxide (DMSO) in a specific ratio. This unique formulation can greatly improve the doubling pollen shedding rate and fruit setting rate of haploid plants, thereby significantly improving the production efficiency of double haploid (DH) plants. The application of this invention effectively solves the problem of low doubling efficiency of haploid embryos in traditional colchicine treatment methods, providing a highly efficient and practical haploid breeding technology solution for the field of plant breeding, thus significantly improving the overall efficiency of haploid breeding technology. Detailed Implementation

[0027] The present invention provides a haploid doubling composition comprising colchicine, DMSO and sorbitol.

[0028] This invention has experimentally demonstrated that sorbitol, when used in combination with colchicine and DMSO in a specific ratio, can significantly increase the haploid doubling rate, thereby greatly improving the haploid doubling pollen rate and fruit setting rate.

[0029] The present invention also provides the application of the above-mentioned haploid doubling composition in improving the haploid doubling efficiency of plant embryos or in plant haploid breeding.

[0030] In this invention, when the haploid doubling composition is used to improve the haploid doubling efficiency of plant embryos, the plant embryos can be inoculated into a culture medium containing the haploid doubling composition to improve the haploid doubling efficiency of plant embryos; when the haploid doubling composition is used in plant haploid breeding, haploid plants can be induced by anther culture or other tissue culture techniques, and then the haploid plants can be treated with the haploid doubling composition of this invention to double their chromosome sets, thereby obtaining homozygous diploids and completing plant haploid breeding; in this invention, the plant is preferably corn, rice, potato, sugar beet or wheat.

[0031] The present invention also provides a haploid doubling culture medium containing the above-mentioned haploid doubling composition; preferably, the final concentration of colchicine in the haploid doubling culture medium is 0.01-2 g / L, more preferably 0.05-0.5 g / L; the final volume percentage concentration of DMSO is 1-5%, more preferably 2-3%; and the final concentration of sorbitol is 0.5-20 g / L, more preferably 1-10 g / L, and most preferably 3-8 g / L; based on the dosage range of each reagent described in the present invention, the optimal haploid doubling effect can be achieved.

[0032] In this invention, the haploid doubling culture medium further includes basal culture medium salt components, sucrose, agar, and water. The basal culture medium salt components mentioned in this invention refer to a series of inorganic salt compounds used to prepare the basal culture medium. These are the core components of the culture medium, providing the inorganic nutrients necessary for the growth of microorganisms or cells, including macroelements such as nitrogen, phosphorus, potassium, sulfur, magnesium, and calcium, and microelements such as iron, zinc, manganese, and copper. They are usually provided in dry powder form and need to be accurately weighed according to a specific formula ratio and dissolved in water before use to prepare the required culture medium. The preparation ratio and types of these salt components will vary according to the specific needs of different cultured organisms to meet their specific growth and metabolic conditions.

[0033] In this invention, preferably, the salt components of the basal culture medium are basal salts of 1 / 2 MS culture medium or MS culture medium; preferably, the final concentration of the basal culture medium salt components in the haploid doubling culture medium is 3-5 g / L; the final concentration of sucrose in the haploid doubling culture medium is 10-50 g / L; the final concentration of agar in the haploid doubling culture medium is 5-10 g / L; preferably, the pH of the haploid doubling culture medium is 5.5-7.

[0034] The present invention also provides a method for doubling haploid embryos, comprising the following steps:

[0035] The plant embryos were inoculated into the above-mentioned haploid doubling medium and induced to obtain haploid embryos.

[0036] In one specific embodiment of the present invention, the plant embryo is a maize haploid embryo.

[0037] In this invention, the maize haploid embryo is a maize haploid embryo harvested 12 to 20 days after pollination, after hybridization of a haploid inducing line as the male parent and the induced material, specifically a maize haploid embryo harvested 18 days after pollination.

[0038] In the method provided by the present invention, the parent haploid inducing line can be selected from any of the haploid inducing lines with the R1nj label, such as CAU5.

[0039] In the method provided by the present invention, the following steps are preferably adopted:

[0040] 1) First, obtain germplasm with high-frequency chemical doubling, that is, use chemical agents, such as colchicine, to treat inbred lines that are easy to double, use this material as the induced material as the female parent, and use haploid inducible lines carrying the R1nj marker as the male parent for artificial pollination and hybridization.

[0041] 2) On days 12 to 20 after pollination, the young embryos of the hybrid ears were removed and inoculated into the culture medium containing the maize haploid doubling agent and the corresponding control culture medium without the doubling agent for doubling treatment.

[0042] 3) Select haploid embryos with colorless scutellar color from the embryos after 12h to 72h of treatment and carry out seedling culture;

[0043] 4) After transplanting the seedlings obtained from the seedling cultivation, self-pollinate them to obtain haploid maize DH seeds with doubled chromosomes.

[0044] In the method provided by this invention, to ensure that the flowering periods of the parent plants coincide, the female plant can be planted once, and the male plant can be sown multiple times (e.g., 23 times) around the sowing period of the female plant. During the artificial pollination process, the female plant is emasculated (the filaments are removed) during its flowering period, the female ears are strictly bagged, and pollen from the male plant is used for artificial over-pollination, with the pollination time recorded.

[0045] In the method provided by this invention, the culture conditions for the doubling treatment are full light, temperature of about 26°C, and humidity of about 60%.

[0046] In the method provided by this invention, the seedling cultivation time can be 2 days or 7 days, specifically 5 days.

[0047] In the method provided by this invention, the cultivation conditions for seedling cultivation are an alternating 16-hour light period / 8-hour dark period, a temperature of about 26°C, and a humidity of about 60%.

[0048] In the method provided by the present invention, the seedlings obtained from the seedling cultivation are transplanted to the field. Before transplanting, the seedlings can be hardened off by acclimatization. Specifically, after the normal seedlings grow to 23 leaves and one bud, they are transplanted into nutrient pots filled with peat moss to acclimatize and harden off. After they grow to 5 leaves and one bud, they are transplanted to the field.

[0049] In the method provided by the present invention, the maize haploid embryo is a maize haploid embryo harvested 12 to 20 days after pollination after hybridization with a haploid inducing line as the male parent and the induced material, specifically a maize haploid embryo harvested 18 days after pollination.

[0050] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] The materials used in the embodiments of this invention are sourced from the following sources:

[0052] The maize variety Zhengdan 958 involved in the haploid in this embodiment of the invention has the approval number Guoshenyu 20000009. Non-patent literature describing this variety includes "Wu PH, Li HC, Ren J, et al. Mapping of maternal QTLs for in vivo haploid induction rate in maize (Zea mays L.). Euhytica, 2014, 196(3): 413-421", which can be obtained by the public from Beijing Denong Seed Industry Co., Ltd. This material is only used to repeat the relevant experiments of this invention and cannot be used for other purposes.

[0053] The maize (Zea mays L.) haploid inducible line CAU5 in this invention embodiment has the R1nj marker. It was provided by Professor Chen Shaojiang of China Agricultural University and disclosed in the literature "Jiao Yanyan. Evaluation and contemporary genetic effects of maize haploid inducible lines. [D]. China Agricultural University, 2017." The public can obtain this material from the applicant. This material is only used to repeat the relevant experiments of this invention and cannot be used for other purposes.

[0054] In the embodiments of this invention, both sucrose and agarose are products of Sinopharm Shanghai Trial.

[0055] The sorbitol, dimethyl sulfoxide, and colchicine used in the embodiments of this invention were purchased from Beijing Boyouhang Biotechnology Co., Ltd., and the MS salt was purchased from Shanghai Yuhan Biotechnology Co., Ltd.

[0056] Example 1

[0057] A culture medium containing 4.33 g / L MS salt, 30 g / L sucrose, 7.5 g / L agar, 0.1 g / L colchicine, 2% (v / v) DMSO, 1 g / L sorbitol, and the remainder water, with a pH of 5.8, was prepared.

[0058] Example 2

[0059] A culture medium containing 4.33 g / L MS salt, 30 g / L sucrose, 7.5 g / L agar, 0.1 g / L colchicine, 2% (v / v) DMSO, 5 g / L sorbitol, and the remainder water, with a pH of 5.8, was prepared.

[0060] Example 3

[0061] A culture medium containing 4.33 g / L MS salt, 30 g / L sucrose, 7.5 g / L agar, 0.1 g / L colchicine, 2% (v / v) DMSO, 10 g / L sorbitol, and the remainder water, with a pH of 5.8, was prepared.

[0062] Example 4

[0063] Zhengdan 958 was used as the female parent (the induced material), and the inducing line CAU5 was used as the male parent to induce haploids. The male parent CAU5 was planted in two phases: the first phase was sown on April 25th, and the second phase on April 30th. The female parent was sown at the same time as the first phase of the male parent to ensure simultaneous flowering. The stamens of the female parent were uniformly cut, and strict emasculation was performed. Overpollination was carried out using the male parent inducing line, and the pollination time was recorded uniformly. Embryos 18 days after pollination (denoted as DAP15 days) were used as material and inoculated into the culture medium provided in Example 1 for in vitro embryo culture for 24 hours. The culture conditions were full light, a temperature of approximately 26°C, and a humidity of approximately 60%.

[0064] CAU5 embryos carry the R1-nj marker. After 24 hours of embryo culture, the scutellum of heterozygous diploid embryos appears purplish-red due to the presence of the R1-nj marker. Haploid and double haploid (DH) embryos contain only one set of chromosomes from the maternal material and do not carry the R1-nj marker; therefore, the scutellum of haploid embryos appears colorless. Haploid embryos (with colorless scutellums) are selected based on scutellum color and inoculated onto seedling culture medium for seedling culture.

[0065] The seedling culture medium contains 2.16 g / L MS salt, 30 g / L sucrose, 7.5 g / L agar, and has a pH of 5.8.

[0066] Cultivation conditions: full sunlight, temperature around 26℃, humidity around 60%. Place in a cultivation room for 7 days to cultivate seedlings. Then, harden off outdoors. Transplant to the field when the seedlings have 3-5 leaves. When the haploid plants emerge from the tassels and silks, strictly bag the female ears. After the haploids shed pollen, strictly self-pollinate until maturity and harvest.

[0067] Experimental Example

[0068] 1. Induction of maize haploid embryos

[0069] Zhengdan 958 was used as the female parent (the induced material), and the inducing line CAU5 was used as the male parent to induce haploids. The male parent CAU5 was planted in two phases: the first phase was sown on April 25th, and the second phase on April 30th. The female parent was sown at the same time as the first phase of the male parent to ensure simultaneous flowering. The stamens of the female parent were uniformly removed, and strict emasculation was performed. Overpollination was conducted using the male parent inducing line, and the timing of each pollination was recorded. Embryos 18 days after pollination (referred to as DAP15 days) were used for in vitro embryo culture.

[0070] 2. Identification and Doubling of Haploid Immature Embryos in Maize

[0071] The embryos obtained in step 1, 18 days after pollination, were inoculated into the following culture media for treatment:

[0072] The doubled differential medium with a CK concentration of 0 mg / L specifically contained MS salt at a concentration of 4.33 g / L, sucrose at a concentration of 30 g / L, agar at a concentration of 7.5 g / L, sorbitol at a concentration of 0 mg / L, and water as the remainder, with a pH of 5.8.

[0073] The doubled differential medium with a CK+A1 content of 1 g / L specifically contains 4.33 g / L MS salt, 30 g / L sucrose, 7.5 g / L agar, 1 g / L sorbitol, and the remainder is water, with a pH of 5.8.

[0074] The doubled differential medium with a CK+A2 content of 5 g / L specifically contains 4.33 g / L MS salt, 30 g / L sucrose, 7.5 g / L agar, 5 g / L sorbitol, and the remainder is water, with a pH of 5.8.

[0075] The doubled differential medium with CK+A3 content of 10 g / L specifically contains MS salt at 4.33 g / L, sucrose at 30 g / L, agar at 7.5 g / L, sorbitol at 10 g / L, and the remainder is water, with a pH of 5.8.

[0076] K1: Doubled differential medium with a concentration of 0 mg / L, specifically containing 4.33 g / L MS salt, 30 g / L sucrose, 7.5 g / L agar, 0.1 g / L colchicine, 2% (v / v) DMSO, 0 mg / L sorbitol, and the remainder water, with a pH of 5.8.

[0077] The doubled differential medium with a K1+A1 content of 1 g / L specifically contains 4.33 g / L MS salt, 30 g / L sucrose, 7.5 g / L agar, 0.1 g / L colchicine, 2% (v / v) DMSO, 1 g / L sorbitol, and the remainder is water, with a pH of 5.8.

[0078] The doubled differential medium with a K1+A2 content of 5 g / L specifically contains 4.33 g / L MS salt, 30 g / L sucrose, 7.5 g / L agar, 0.1 g / L colchicine, 2% (v / v) DMSO, 5 g / L sorbitol, and the remainder is water, with a pH of 5.8.

[0079] The doubled differential medium with K1+A3 content of 10 g / L specifically contains MS salt at 4.33 g / L, sucrose at 30 g / L, agar at 7.5 g / L, colchicine at 0.1 g / L, DMSO at 2% (v / v), sorbitol at 10 g / L, and the remainder is water, with a pH of 5.8.

[0080] The culture time for each treatment group was 24 hours. Culture conditions included full light, a temperature of approximately 26℃, and a humidity of approximately 60%. CAU5 embryos carry the R1-nj marker. After 24 hours of embryo culture, the scutellum of heterozygous diploid embryos appeared purplish-red due to carrying the R1-nj marker. Haploid embryos and double haploid (DH) embryos contained only one set of chromosomes from the maternal material and did not contain the R1-nj marker; therefore, the scutellum of haploid embryos was colorless. Haploid embryos (with colorless scutellums) were selected based on scutellum color and inoculated onto seedling culture medium for seedling culture.

[0081] The seedling culture medium consisted of 1 / 2 MS solid medium, specifically containing 2.16 g / L MS salt, 30 g / L sucrose, 7.5 g / L agar, and a pH of 5.8.

[0082] The cultivation conditions were full light, a temperature of approximately 26℃, and a humidity of approximately 60%. Seedlings were placed in a cultivation room for 7 days. Afterward, they were hardened off outdoors until they reached the 3-5 leaf stage, at which point they were transplanted into the field. When the haploid plants began to tassel and silk, the female ears were strictly bagged. After pollen shedding, strict self-pollination was performed. After maturity and harvest, key indicators such as haploid pollen shedding rate, seed setting rate, average seed number, and DH productivity were statistically analyzed to evaluate the haploid doubling efficiency of each treatment. The seeds obtained from haploid self-pollination are shown.

[0083] The grains were double haploids (DH). Specific results are shown in Table 1.

[0084] Pollen shedding rate = (Number of haploid plants with loose pollen / Total number of haploid plants) × 100%

[0085] Fruit set rate = (Number of fruit-bearing haploid plants / Number of pollen-shedding haploid plants) × 100%

[0086] DH productivity = Number of fruit-bearing haploid plants / Total number of haploid plants × 100%.

[0087] Table 1. Effects of different sorbitol contents on doubling of haploid embryos.

[0088] deal with Number of pollen plants Number of plants without pollen Total number of plants loose powder rate Number of fruit-bearing plants Average seed setting rate DH productivity CK 29 121 150 19.33％ 14 48.28％ 9.33％ CK+A1 31 119 150 20.67％ 13 41.94％ 8.67％ CK+A2 36 114 150 24.00％ 15 41.67％ 10.00％ CK+A3 34 116 150 22.67％ 12 35.29％ 8.00％ K1 86 64 150 57.33％ 56 65.12％ 37.33％ K1+A1 93 57 150 62.00％ 78 83.87％ 52.00％ K1+A2 97 53 150 64.67％ 85 87.63％ 56.67％ K1+A3 91 59 150 60.67％ 75 82.42％ 50.00％

[0089] As demonstrated by the above embodiments, this invention provides an effective method for doubling haploid embryos. Through specific culture media and treatment conditions, it significantly improves the doubling efficiency of haploid embryos. Experimental results show that the treatment groups (K1+A1, K1+A2, K1+A3) treated with culture media containing colchicine and different concentrations of sorbitol significantly improved pollen shedding rate, seed setting rate, and DH productivity compared to the control group (CK, CK+A1, CK+A2, CK+A3). In particular, the K1+A2 treatment group achieved the highest values ​​in pollen shedding rate, seed setting rate, and DH productivity, at 64.67%, 87.63%, and 56.67%, respectively. This indicates that this invention can effectively improve the doubling efficiency of haploid embryos, thereby increasing the yield of double haploid (DH) seeds. The method provided by this invention offers new ideas and practical guidance for maize haploid doubling technology, possessing significant application value and promising prospects for promotion.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A maize haploid doubling culture medium, characterized in that, The haploid doubling culture medium contains a haploid doubling composition, which includes colchicine, DMSO and sorbitol; In the haploid doubling culture medium, the final concentration of colchicine is 0.01~2 g / L; In the haploid doubling culture medium, the final volume percentage concentration of DMSO is 1-5%. In the haploid doubling culture medium, the final concentration of sorbitol is 0.5~20 g / L; The haploid doubling medium also includes basal medium salt components, sucrose, agar, and water.

2. The haploid doubling culture medium according to claim 1, characterized in that, The salt components of the basal culture medium are either 1 / 2 MS medium basal salts or MS medium basal salts.

3. The haploid doubling culture medium according to claim 1, characterized in that, In the haploid doubling medium, the final concentration of the salt components of the basal medium is 3~5 g / L; In the haploid doubling culture medium, the final concentration of sucrose is 10~50g / L; In the haploid doubling medium, the final concentration of agar is 5-10 g / L.

4. The haploid doubling culture medium according to claim 1, characterized in that, The pH of the haploid doubling medium is 5.5-7.

5. A method for doubling haploid embryos, characterized in that, Includes the following steps: The plant embryos are inoculated into the haploid doubling medium as described in any one of claims 1 to 4 and induced to obtain haploid embryos. The induction culture time is 10~30h.