Haploid doubling composition and haploid immature embryo doubling method

Abstract

The invention provides a haploid doubling composition and a haploid immature embryo doubling method, and belongs to the technical field of crop breeding. According to the method, sorbitol, colchicine and dimethyl sulfoxide (DMSO) are compounded for use, so that the effect of remarkably increasing the doubling rate when the haploid plant material is treated is achieved, and the problem of low haploid immature embryo doubling efficiency in a traditional colchicine treatment method is effectively solved. According to the unique formula provided by the invention, the doubled powder scattering rate and the maturing rate of haploid plants can be greatly improved, and then the production efficiency of double haploids (DH) is greatly improved. Through the application of the invention, an efficient and practical haploid breeding technical scheme can be provided for the field of plant breeding, so that the overall efficiency of the haploid breeding technology is remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of crop breeding, and in particular to a haploid doubling composition and a haploid immature embryo doubling method. Background Art

[0002] Haploid breeding technology includes four parts: haploid induction, identification, doubling and DH management. Among them, the induction and identification of haploids are determined by the haploid induction line and the 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 of haploid breeding. The doubling efficiency of haploids is the key link in haploid breeding, that is, the production efficiency of DH is one of the important factors affecting the efficiency of haploid breeding.

[0003] At present, the most widely used haploid doubling methods are the bud immersion method and the haploid young embryo tissue culture doubling method. Compared with the bud immersion method, the doubling efficiency of haploid young embryos is higher, and it is one of the best ways to produce DH in factories. Efficient doubling methods also need to be combined with efficient doubling reagents. Currently, the commonly used haploid doubling reagents include colchicine and herbicides. Among them, the doubling effect of colchicine is significantly better than that of herbicides and is widely used. The doubling of haploids includes two aspects: doubling of male spikes and doubling of female spikes. The doubling of male spikes directly determines the pollen shedding rate after the haploid doubling treatment, and the doubling of female spikes determines the fruit setting rate. Haploids need to double both male and female spikes at the same time to obtain DH normally. Therefore, ensuring the high-frequency pollen shedding rate and fruit setting rate of haploids is the key to improving the efficiency of haploid doubling.

[0004] Chinese patent CN105918116A discloses a method for doubling plant haploids, comprising soaking the radicle part of the treated plant haploid sprouts in a mixture of 0.02%-0.05% colchicine and 2% dimethyl sulfoxide by mass, the treatment time being 18-36 hours and the temperature being 14-18°C.

[0005] However, the doubling effect of the plant haploid doubling method in the prior art still needs to be improved. Summary of the invention

[0006] The object of the present invention is to provide a haploid doubling composition and a haploid immature embryo doubling method, which have higher doubling efficiency.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0009] The present invention also provides the use of the haploid doubling composition in improving the haploid doubling efficiency of plant immature 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, wherein the haploid doubling culture medium contains the haploid doubling composition.

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

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

[0014] In the haploid doubling medium, the final concentration of sorbitol is 0.5-20 g / L.

[0015] Preferably, the haploid doubling medium further comprises a basal medium salt component, sucrose, agar and water.

[0016] In the present invention, the basic culture medium salt components refer to a series of inorganic salt compounds used to prepare the basic culture medium, which are the core components of the culture medium. They provide inorganic nutrients necessary for the growth of microorganisms or cells, such as macroelements such as nitrogen, phosphorus, potassium, sulfur, magnesium, calcium, and trace elements such as iron, zinc, manganese, and copper. They are usually provided in the form of dry powder, and need to be accurately weighed according to a specific formula ratio before use, and dissolved in water to prepare the required culture medium. The preparation ratio and type of these salt components will vary according to the specific needs of different culture objects to meet their specific conditions for growth and metabolism.

[0017] Preferably, the basal culture medium salt component is 1 / 2MS culture medium basal salt or MS culture medium basal salt.

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

[0019] In the haploid doubling 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 medium is 5.5-7.

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

[0023] Inoculating the plant embryos into the above haploid doubling medium for induction culture to obtain haploid embryos;

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

[0025] Beneficial effects of the present invention:

[0026] The present invention, by mixing sorbitol, colchicine and dimethyl sulfoxide (DMSO) in a specific ratio, realizes the effect of significantly improving the doubling rate when processing haploid plant materials. This unique formula can significantly improve the doubling powder rate and fruit setting rate of haploid plants, and then greatly improve the production efficiency of double haploid (DH). Through the application of the present invention, the problem of low doubling efficiency of haploid young embryos in traditional colchicine treatment methods can be effectively solved, and a kind of efficient and practical haploid breeding technology scheme is provided for the field of plant breeding, thereby significantly improving the overall efficiency of haploid breeding technology. DETAILED DESCRIPTION

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

[0028] The present invention has verified through experiments that the sorbitol can act together with colchicine and DMSO in a specific ratio to significantly improve the haploid treatment doubling rate, and greatly improve the haploid doubling powder shedding rate and fruit setting rate.

[0029] The present invention also provides the use of the haploid doubling composition in improving the haploid doubling efficiency of plant immature embryos or in plant haploid breeding.

[0030] In the present invention, when the haploid doubling composition is used to improve the haploid doubling efficiency of plant immature embryos, the plant immature embryos can be inoculated into a culture medium containing the haploid doubling composition to achieve improved haploid doubling efficiency of plant immature embryos; when the haploid doubling composition is used in plant haploid breeding, haploid plants can be induced by anther in vitro culture or other tissue culture techniques, and then the haploid doubling composition of the present invention is used to treat the haploid plants to double their chromosome sets and obtain homozygous diploids, thereby completing plant haploid breeding; in the present invention, the plants are preferably corn, rice, potatoes, beets or wheat.

[0031] The present invention also provides a haploid doubling medium, which contains the above-mentioned haploid doubling composition; preferably, in the haploid doubling medium, the final concentration of colchicine is 0.01-2 g / L, and more preferably 0.05-0.5 g / L; the final volume percentage concentration of DMSO is 1-5%, and more preferably 2-3%; the final concentration of sorbitol is 0.5-20 g / L, and 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 the present invention, the haploid doubling medium also includes a basic medium salt component, sucrose, agar and water. The basic medium salt component of the present invention refers to a series of inorganic salt compounds used to prepare the basic medium, which is the core component of the culture medium. They provide inorganic nutrients necessary for the growth of microorganisms or cells, such as macro elements such as nitrogen, phosphorus, potassium, sulfur, magnesium, calcium, and trace elements such as iron, zinc, manganese, and copper. They are usually provided in the form of dry powder and need to be accurately weighed according to a specific formula ratio before use, and dissolved in water to prepare the required culture medium. The preparation ratio and type of these salt components will vary according to the specific needs of different culture objects to meet their specific conditions for growth and metabolism.

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

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

[0035] The plant immature embryos are inoculated into the above haploid doubling medium for induction culture to obtain haploid immature embryos.

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

[0037] In the present invention, the maize haploid embryos are maize haploid embryos peeled off 12 to 20 days after pollination after hybridization with a haploid induction line as the male parent and an induced material, and specifically can be maize haploid embryos peeled off 18 days after pollination.

[0038] In the method provided by the present invention, the male parent of the haploid induction line can be selected from any one of the haploid induction lines having the R1nj marker, 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 an inbred line that is easy to double, use this material as the induced material, as the female parent, and use the haploid induced line carrying the R1nj marker as the male parent for artificial pollination hybridization;

[0041] 2) 12 to 20 days after pollination, the hybrid ears are taken and young embryos are peeled off, and the embryos are inoculated into the culture medium containing the maize haploid doubling agent and the corresponding control culture medium not containing the doubling agent for doubling treatment;

[0042] 3) Selecting haploid embryos with colorless scutellum from the embryos treated for 12 to 72 hours for seedling culture;

[0043] 4) After the seedlings obtained from the seedling cultivation are planted, they are self-pollinated to obtain haploid chromosome-doubled corn DH seeds.

[0044] In the method provided by the present invention, in the artificial pollination hybridization, to ensure that the flowering periods of the parents meet, the female parent can be planted for one period, and the male parent can be sown for multiple periods (for example, 23 periods) around the sowing period of the female parent. In the artificial pollination hybridization, the female parent is emasculated (the filaments are cut off) during the flowering period, the female ears are strictly bagged, the pollen of the male parent is taken for artificial over-pollination, and the pollination time is recorded.

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

[0046] In the method provided by the present invention, the time for the seedling culture can be 2 days to 7 days, specifically 5 days.

[0047] In the method provided by the present invention, the culture conditions for the seedling culture are 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 by the seedling cultivation are planted in the field. Before planting, the seedlings can be hardened and acclimatized. Specifically, after the normal embryo seedlings grow to 23 leaves and one heart, they are transplanted into a nutrient pot filled with peat soil for hardening and acclimatization. After they grow to 5 leaves and one heart, they are planted in the field.

[0049] In the method provided by the present invention, the maize haploid immature embryo is a maize haploid immature embryo peeled off 12 to 20 days after pollination after hybridization with a haploid induction line as the male parent and an induced material, and specifically can be a maize haploid immature embryo peeled off 18 days after pollination.

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

[0051] The sources of materials used in the embodiments of the present invention are as follows:

[0052] The haploid maize variety Zhengdan 958 involved in the embodiment of the present invention has an approval number of Guoshenyu 20000009. The non-patent literature recording 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.). Euphytica, 2014, 196 (3): 413-421", etc., which can be obtained by the public from Beijing Denong Seed Co., Ltd. This material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

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

[0054] The sucrose and agarose in the embodiments of the present invention are both products of National Medicine Shanghai Trial.

[0055] Sorbitol, dimethyl sulfoxide, and colchicine in the examples of the present invention were purchased from Beijing Boyuhang Biotechnology Co., Ltd., and 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 value 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 value 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 value of 5.8 was prepared.

[0062] Example 4

[0063] Zhengdan 958 was used as the female parent (induced material), and the induction 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 25, and the second phase was sown on April 30; the sowing period of the female parent was the same as that of the first phase of the male parent so that the flowering period coincided. The female parent uniformly cut the filaments, strictly emasculated, and over-pollinated with the male parent induction line, and the unified pollination time was recorded at the same time. The young embryos 18 days after pollination (recorded as DAP15 days) were used as materials and inoculated into the culture medium provided in Example 1, and the young embryos were cultured in vitro for 24 hours. The culture conditions were full light, temperature around 26°C, and humidity around 60%.

[0064] CAU5 has the R1-nj marker. After 24 hours of embryo culture, the scutellum of the heterozygous diploid embryos appears purple-red due to the R1-nj marker. Haploid embryos and double haploid (DH) embryos contain only one set of chromosomes of the maternal material and do not contain the R1-nj marker, so the scutellum of the haploid embryos appears colorless. Haploid embryos (scutellum of the embryos appears colorless) are selected according to the color of the scutellum and inoculated onto the seedling medium for seedling culture.

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

[0066] The culture conditions are full light, temperature around 26℃, and humidity around 60%. Place in the culture room and culture for 7 days. Then carry out outdoor seedling hardening, and transplant to the field when the seedlings are 3-5 leaves. When the haploid plants are masculine and silk, the female ears are strictly bagged, and the haploids are strictly self-pollinated after they shed pollen, and harvested until mature.

[0067] Experimental example

[0068] 1. Induction of Haploid Immature Embryos in Maize

[0069] Zhengdan 958 was used as the female parent (induced material) and the induction 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 25, and the second phase was sown on April 30; the sowing period of the female parent was the same as that of the first phase of the male parent so that the flowering period would coincide. The female parent uniformly cut the filaments, strictly emasculated, and over-pollinated with the male parent induction line, and the uniform pollination time was recorded. The young embryos 18 days after pollination (recorded as DAP15 days) were used as materials for in vitro culture of young embryos.

[0070] 2. Identification and doubling of maize haploid embryos

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

[0072] The doubled identification medium with a CK content of 0 mg / L specifically contains 4.33 g / L of MS salt, 30 g / L of sucrose, 7.5 g / L of agar, 0 mg / L of sorbitol, and the rest is water, with a pH value of 5.8.

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

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

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

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

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

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

[0079] The doubled identification medium with a K1+A3 content of 10 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, 10 g / L sorbitol, and the rest is water, with a pH value of 5.8.

[0080] The culture time of each treatment group is 24 hours. The culture conditions are full light, temperature of about 26°C, and humidity of about 60%. CAU5 has an R1-nj marker. After 24 hours of culturing the young embryos, the scutellum of the heterozygous diploid young embryos appears purple-red due to the R1-nj marker. The haploid young embryos and double haploid (DH) young embryos only contain one set of chromosomes of the maternal material and do not contain the R1-nj marker. Therefore, the scutellum of the haploid young embryos appears colorless. The haploid young embryos (the scutellum of the young embryos appears colorless) are selected according to the color of the scutellum and inoculated onto the seedling medium for seedling culture.

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

[0082] The culture conditions are full light, temperature around 26°C, and humidity around 60%. Place in the culture room and culture for 7 days. Then carry out outdoor seedling hardening, and transplant to the field when the seedlings are 3-5 leaves. When the haploid plants are tasseled and silked, the female ears are strictly bagged, and the haploids are strictly self-pollinated after they shed pollen. After mature harvest, the key indicators such as haploid pollen shedding rate, fruit setting rate, average fruit setting number DH productivity, etc. are counted to evaluate the doubling efficiency of haploids in each treatment. Seeds obtained from haploid self-pollination.

[0083] The specific results are shown in Table 1.

[0084] Pollen shedding rate = number of haploid plants shedding pollen / total number of haploid plants × 100%

[0085] Fruiting rate = number of fruiting haploid plants / number of haploid plants with loose pollen × 100%

[0086] DH productivity = number of fruiting haploid plants / total number of haploid plants × 100%.

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

[0088] deal with Number of loose pollen plants Number of plants without pollen Total number of plants Powder loosening rate Number of fruiting plants Average fruit 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 can be seen from the above embodiments, the present invention provides an effective method for doubling haploid immature embryos, which significantly improves the doubling efficiency of haploid immature embryos through specific culture medium and treatment conditions. The experimental results show that the culture medium treatment groups (K1+A1, K1+A2, K1+A3) added with colchicine and different concentrations of sorbitol have significantly improved the powder shedding rate, fruit setting rate and DH productivity compared with the control group (CK, CK+A1, CK+A2, CK+A3). In particular, the K1+A2 treatment group reached the highest values ​​in powder shedding rate, fruit setting rate and DH productivity, which were 64.67%, 87.63% and 56.67% respectively. This shows that the present invention can effectively improve the doubling efficiency of haploid immature embryos, thereby increasing the yield of double haploid (DH) seeds. The method provided by the present invention provides new ideas and practical guidance for corn haploid doubling technology, and has important application value and promotion prospects.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A haploid doubling composition, characterized in that: These include colchicine, DMSO, and sorbitol.

2. Use of the haploid doubling composition according to claim 1 in improving the haploid doubling efficiency of plant immature embryos or in plant haploid breeding.

3. The use according to claim 2, characterized in that: The plant is corn, rice, potato, sugar beet or wheat.

4. A haploid doubling medium, characterized in that: The haploid doubling medium contains the haploid doubling composition according to claim 1.

5. The haploid doubling medium according to claim 4, characterized in that: In the haploid doubling medium, the final concentration of colchicine is 0.01-2 g / L; In the haploid doubling medium, the final volume percentage concentration of DMSO is 1-5% In the haploid doubling medium, the final concentration of sorbitol is 0.5-20 g / L.

6. The haploid doubling medium according to claim 4 or 5, characterized in that: The haploid doubling medium also includes basal medium salt components, sucrose, agar and water.

7. The haploid doubling medium according to claim 6, characterized in that: The basic culture medium salt component is 1 / 2MS culture medium basic salt or MS culture medium basic salt.

8. The haploid doubling medium according to claim 6, characterized in that: In the haploid doubling medium, the final concentration of the salt component of the basal medium is 3-5 g / L; In the haploid doubling medium, the final concentration of sucrose is 10-50 g / L; In the haploid doubling medium, the final concentration of agar is 5-10 g / L.

9. The haploid doubling medium according to claim 6, characterized in that: The pH of the haploid doubling medium is 5.5-7.

10. A method for doubling haploid immature embryos, characterized in that: The following steps are involved: Inoculating the plant embryos into the haploid doubling medium according to any one of claims 4 to 9 for induction culture to obtain haploid embryos; The induction culture time is 10 to 30 hours.