Method for obtaining plant high-frequency embryogenesis material

Abstract

The invention belongs to the technical field of plant tissue culture and genetic transformation, and particularly relates to a method for obtaining a plant high-frequency embryogenesis material. The method comprises the following steps: inoculating a plant explant to a callus induction culture medium for culturing; carrying out continuous subculture on the callus for 4-6 times; transferring the embryogenic callus growing after the subculture to a budding culture medium for culture, and inducing the embryogenic callus on the budding culture medium to form adventitious buds; transferring the adventitious buds to a rooting culture medium, and culturing until rooting; and transplanting the plant with the root system, and culturing until the plant is mature and fruitful to obtain the high-frequency embryogenesis material. According to the method provided by the invention, a mature embryo of a low-frequency embryogenesis material can be inoculated on an induction culture medium, embryogenic calluses are formed through induction, the formed embryogenic calluses are regenerated and induced into seedlings, and a high-frequency embryogenesis material under the genotype background can be obtained through one-time or multiple-time repeated screening.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture and genetic transformation, and in particular relates to a method for obtaining plant high-frequency embryogenesis material. Background Art

[0002] At present, genetic transformation systems have been established for many crops, and gene editing breeding can be carried out. However, due to genotype restrictions, only a few varieties / lines of these crops can be used for genetic transformation, and the varieties widely promoted and applied in production are often difficult to directly genetically transform. The problem of genotype restriction in the genetic transformation process is common in various crops, which greatly restricts gene editing and transgenic breeding, and has become one of the biggest bottlenecks restricting gene editing and transgenic breeding. This is mainly because most breeding materials lack renewable genes, and the callus induced by their explants cannot undergo embryogenesis or has a very low embryogenesis rate. After tissue culture, the regeneration rate is very low, and seedlings cannot be formed, and seeds with successful genetic transformation cannot be obtained. In order to widely promote the application of genetic transformation or gene editing breeding technology in crop breeding, the problem of genotype restriction in crop genetic breeding must be solved.

[0003] At present, the method of overexpressing embryonic development genes through transgenic technology can effectively solve the genotype restriction problem in crop genetic breeding. However, the method of overexpressing embryonic development genes through transgenic technology will introduce foreign genes, involving complex transgenic operations and difficult to operate. Summary of the invention

[0004] In order to solve the problem that the method of overexpressing embryo development genes through transgenic technology in existing genetic transformation technology will introduce foreign genes, involve complex transgenic operations, and have great difficulty in operation, the present invention provides a method for obtaining plant high-frequency embryogenesis material.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0006] The present invention provides a method for obtaining a plant high-frequency embryogenesis material, comprising the following steps:

[0007] The plant explants are sterilized, washed and inoculated on callus induction medium to obtain callus tissue, and the callus tissue is subcultured 4 to 6 times, with a subculture interval of 15 to 20 days.

[0008] The embryonic callus that grows after 4 to 6 subcultures is transferred to a budding medium for culture, and the embryonic callus is induced to form adventitious buds on the budding medium. Among them, the formation of embryonic callus is a key step in tissue culture regeneration. The formed embryonic callus is selected and transferred to the budding medium, and the callus that cannot form embryonic callus is eliminated.

[0009] The adventitious shoots were transferred to rooting medium and cultured until roots formed.

[0010] The rooted plants are transplanted and cultured until they are mature and strong, thereby obtaining the high-frequency embryogenesis material.

[0011] The present invention provides a method for obtaining plant embryogenic materials, which can inoculate mature embryos of low-frequency embryogenic materials on an induction medium to induce the formation of embryonic calli, and induce the formed embryonic calli to regenerate into seedlings. After one or more repeated screenings, high-frequency embryogenic materials under the genotype background can be obtained. The present invention provides a method for obtaining plant embryogenic materials that does not require overexpression of regeneration genes. Plant explants are used as materials, and callus tissue is induced and cultured, and then high-frequency embryogenic materials suitable for plant genetic transformation are obtained through continuous tissue culture screening. The method for obtaining plant embryogenic materials provided by the present invention can not only effectively solve the genotype restriction problem in genetic breeding, but also has the advantages of being relatively simple and easy to operate, no introduction of exogenous genes, no complicated transgenic operations, and no restrictions from relevant laws and regulations, and can effectively improve the embryogenesis rate.

[0012] At the same time, the method for obtaining plant embryogenic materials provided by the present invention can effectively screen out individuals with high-frequency embryogenic ability in crop varieties and retain them, thereby continuously improving the embryogenicity rate in the variety.

[0013] Preferably, the plant explants include any one of mature embryos, immature embryos and hypocotyls. The callus induced by these materials has strong regeneration ability and can form embryonic callus well.

[0014] Preferably, the conditions for culturing the plant explants on the callus induction medium are: 24°C to 26°C, 13h to 15h of light. More preferably, the conditions for culturing the plant explants on the callus induction medium are: 25°C, 14h of light.

[0015] Preferably, the formula composition of the callus induction medium is: MS solid powder medium 4.44g / L, sucrose 30g / L-50g / L, 2,4-D 0.5mg / L-3mg / L, KT 0.1mg / L-1mg / L, plant gel 2.6g / L-4.0g / L, and the solvent is water. This medium formula can well induce plant materials to form embryonic callus.

[0016] Since the embryogenesis rate of the varieties widely used in production is low and difficult to be used for genetic transformation, the high-frequency embryogenesis materials currently suitable for plant genetic transformation are basically not the varieties widely used in production. For example, Hi-II and B104, which are commonly used for genetic transformation in corn, and Ci846, which are used for genetic transformation in millet, are not the varieties widely used in production. To establish a genetic transformation system for varieties widely used in production, the conditions of the entire plant tissue culture must be optimized, which is often difficult to succeed, and the optimal culture conditions screened by this method cannot be replicated in other varieties.

[0017] The method for obtaining plant embryogenic materials provided by the present invention selects individuals suitable for the culture medium from a certain variety through domestication and screening on the same culture medium, and selects and purifies them, thereby improving the embryogenic rate of the variety and avoiding a large amount of culture medium optimization. The method provided by the present invention successfully increases the embryogenic rate of the varieties Taixuangu 24, Huangmaogu, Jigu 41, and Jigu 39, which are widely promoted and applied in millet production, to more than 40%, so that they can be used for downstream genetic transformation and gene editing, and expand the millet genetic transformation system to multiple production varieties.

[0018] Preferably, the frequency of subculture is once every 15 days.

[0019] Preferably, the embryonic callus is cultured on the budding medium at 24°C to 26°C and 13h to 15h of light. More preferably, the embryonic callus is cultured on the budding medium at 25°C and 14h of light.

[0020] Preferably, the budding medium comprises: 4.44 g / L MS solid powder medium, 30 g / L to 50 g / L sucrose, 0.5 mg / L to 5 mg / L 6-BA, 2.6 g / L to 4.0 g / L plant gel, and the solvent is water. The medium can well induce the embryonic callus of the plant to form adventitious buds.

[0021] Preferably, the conditions for culturing the adventitious buds on the rooting medium are: 24°C to 26°C, 13h to 15h of light per day. More preferably, the conditions for culturing the adventitious buds on the rooting medium are: 25°C, 14h of light per day.

[0022] Preferably, the formula composition of the rooting medium is: MS solid powder medium 2.22g / L, sucrose 30g / L-50g / L, IBA 0.5mg / L-5mg / L, plant gel 2.6g / L-4.0g / L, and the solvent is water. The medium can induce the adventitious buds of plants to quickly grow a developed root system.

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

[0024] 1. The present invention provides a method for obtaining high-frequency embryogenic materials of plants. The present invention provides a method for obtaining high-frequency embryogenic materials of plants by tissue culture screening, without introducing exogenous genes, which can not only effectively solve the genotype restriction problem in genetic breeding, but also has the advantages of being relatively simple and easy to operate, not involving complex transgenic operations, not being restricted by relevant laws and regulations, and being able to effectively improve the embryogenesis rate.

[0025] 2. The method for obtaining high-frequency embryogenic materials of plants provided by the present invention selects individuals suitable for the culture medium from a certain variety through domestication and screening on the same culture medium, and selects and purifies them, thereby improving the embryogenesis rate of the variety and avoiding a large amount of culture medium optimization. At the same time, the method for obtaining high-frequency embryogenic materials of plants provided by the present invention can effectively screen out individuals with high-frequency embryogenesis ability in crop varieties and retain them, thereby continuously improving the embryogenesis rate in the variety.

[0026] 3. The method for obtaining high-frequency embryogenic material provided by the present invention is used to screen out individuals incapable of embryogenesis in a population of a selected variety / line through tissue culture, and individuals with embryogenesis ability are retained. After one or more rounds of tissue culture, the frequency of genes with high-frequency embryogenesis rate in low-frequency embryogenic material can be increased, and the embryogenesis rate of the population can be increased, thereby meeting the needs of genetic transformation.

[0027] 4. The present invention can obtain high-frequency embryogenic materials that are not much different from the original varieties by propagating or rooting the adventitious buds formed after tissue culture-induced embryonic callus.

[0028] 5. Although the method of overexpressing embryo development genes through transgenic technology can also effectively solve the genotype restriction problem in genetic breeding, the overexpression of these embryo development genes often has an unfavorable phenotype, resulting in abnormal plant development, which must be removed by backcrossing after transformation. Therefore, compared with the method of obtaining high-frequency embryogenic materials of plants of the present invention, the method of overexpressing embryo development genes through transgenic technology will lead to problems such as long breeding work cycle and complicated operation. The method of obtaining high-frequency embryogenic materials of plants provided by the present invention does not rely on the genetic transformation system of plants, does not introduce exogenous genes, and can improve low-frequency embryogenic materials.

[0029] 6. The improved material of the present invention can stably maintain the characteristic of high-frequency embryogenesis rate, and can be used to solve the problem of low embryogenesis rate and difficulty in genetic transformation of varieties for production, promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the situation where the mature embryo seeds in Example 1 of the present invention were inoculated on the callus induction medium 15 days later.

[0031] Figure 2 This is the embryonic callus formed on the callus induction medium in Example 1 of the present invention.

[0032] Figure 3 These are clustered buds formed by the embryonic callus in Example 1 of the present invention on the budding medium.

[0033] Figure 4 This is the situation of root induction of clustered buds in Example 1 of the present invention.

[0034] Figure 5 This is the situation where the R1 generation seeds in Example 1 of the present invention are inoculated on the callus induction medium.

[0035] Figure 6 The performance of the high-frequency embryogenic material in the field in Example 1 and Example 5 of the present invention; wherein, Figure 6 A in it is Jingu 51; Figure 6 B in the figure is Jingu 51R1; Figure 6 The C in it is Jigu 39; Figure 6 The D in it is Jigu39R1. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0037] Example 1

[0038] The plant material in this embodiment is millet.

[0039] A method for obtaining plant high-frequency embryogenesis material comprises the following steps:

[0040] (1) Explant disinfection: Millet seeds were used as explants. The material generation used was named R0. They were disinfected with 75% alcohol by volume for 1 min, and then disinfected with 10% sodium hypochlorite solution by volume for 20 min. Finally, the millet seeds were washed 5 times with sterile water, placed on sterile filter paper to absorb excess water on the seed surface, and then inoculated onto callus induction medium to induce callus tissue.

[0041] Among them, the varieties of millet seeds are: Jingu 51, which comes from the College of Agriculture of Shanxi Agricultural University.

[0042] The formula composition of the callus induction medium is: MS solid powder medium 4.44g / L, sucrose 30g / L, 2,4-D2mg / L, KT 0.5mg / L, phytogel 2.6g / L, solvent is water, pH 5.8, high temperature sterilization at 121°C for 20min. Among them, the MS solid powder medium is the MS medium powder purchased from Phytotech.

[0043] (2) Callus induction: The callus induction medium inoculated with the explants was placed in a tissue culture room at 25°C and 14 h light intensity. The callus was subcultured every 15 days or so. The formed callus was subcultured to a new callus induction medium. The browned or water-soaked callus was discarded. Figure 1 shown.

[0044] (3) Formation of embryonic callus: After three subcultures, granular embryonic callus gradually formed. Figure 2 As shown, the formation of embryonic callus is a key step in tissue culture regeneration. The formed embryonic callus is selected and transferred to the budding medium, and the callus that cannot form embryonic callus is eliminated.

[0045] The formula of the germination medium is as follows: 4.44 g / L MS solid powder medium, 30 g / L sucrose, 0.5 mg / L 6-BA, 2.6 g / L phytogel, water as solvent, pH 5.8, and high temperature sterilization at 121°C for 20 min. The MS solid powder medium is the MS medium powder purchased from Phytotech.

[0046] (4) Budding: After the embryonic callus is transferred to the budding medium, adventitious buds will grow from the embryonic callus in about 20 days. Figure 3 The adventitious buds can be further propagated on the budding medium for the next experiment. When the adventitious buds grow to 2 cm, they are transferred to the rooting medium. After about 25 days, slender hair roots will grow. The roots formed are as follows Figure 4 shown.

[0047] The formula of the rooting medium is as follows: 2.22 g / L MS solid powder medium, 30 g / L sucrose, 0.5 mg / L IBA, 2.6 g / L plant gel, water as solvent, pH 5.8, and high temperature sterilization at 121°C for 20 min. The MS solid powder medium is the MS medium powder purchased from Phytotech.

[0048] (5) Transplantation and harvesting: Transplant the seedlings with hairy roots into a moist seedling medium and cover with plastic wrap to keep them moist. After the seedlings gradually adapt, remove the plastic wrap and water them regularly. Maintain a temperature of around 25°C and 14h / 10h of light. After the seedlings bloom and bear fruit, harvest the seeds, which are named the R1 generation.

[0049] Among them, the seedling medium is made of peat soil and vermiculite mixed in a ratio of 7:3.

[0050] It should be noted that the R1 generation seeds are the high-frequency embryogenesis materials obtained.

[0051] (6) Determination of embryogenesis rate of harvested seeds: The harvested R1 generation seeds are inoculated onto callus induction medium using the method described in steps (1) to (3) to induce embryonic callus tissue. The embryogenesis rate of the R1 generation seeds after tissue culture selection is improved, such as Figure 5 shown.

[0052] It should be noted that if the embryogenesis rate of the R1 generation seeds does not meet the application requirements, steps (1) to (5) can be repeated to obtain R2 generation seeds. At this time, the R2 generation seeds are the obtained high-frequency embryogenesis materials.

[0053] Example 2

[0054] A method for obtaining plant high-frequency embryogenesis material comprises the following steps:

[0055] The same as Example 1, except that the variety of millet seeds is Taixuangu 24, which is sourced from the College of Agriculture of Shanxi Agricultural University.

[0056] Example 3

[0057] A method for obtaining plant high-frequency embryogenesis material comprises the following steps:

[0058] The same as Example 1, except that the variety of millet seeds is yellow hair millet, which is sourced from the Millet Research Institute of Shanxi Agricultural University.

[0059] Example 4

[0060] A method for obtaining a plant high-frequency embryogenesis material comprises the following steps:

[0061] The same as Example 1, except that the variety of millet seeds is Jigu 41, which is sourced from the College of Agriculture of Shanxi Agricultural University.

[0062] Example 5

[0063] A method for obtaining plant high-frequency embryogenesis material comprises the following steps:

[0064] The same as Example 1, except that the variety of millet seeds is Jigu 39, which is sourced from the College of Agriculture of Shanxi Agricultural University.

[0065] In order to illustrate the effect of the above method, the embryogenesis rates of the R0 generation seeds, R1 generation seeds, and R2 generation seeds in Examples 1 to 5 were statistically analyzed, and the specific method is as follows:

[0066] The R0 generation seeds, R1 generation seeds, and R2 generation seeds were inoculated onto callus induction medium, and the embryogenesis rate at 60 days was counted. The results are shown in Table 1:

[0067] Table 160 days embryo incidence

[0068] Variety name R0 generation% R1 generation % R2 generation % Jingu 51 62.95±2.93 63.23±6.42 75.48±3.70* Taixuan Valley 24 32.44±6.05 54.98±1.00* / Huangmaogu 9.50±2.10 55.08±6.21** / Jigu41 4.52±2.27 41.22±2.07** / Jigu39 9.13±1.10 59.21±3.00** /

[0069] Note: The data in the table have been repeated three times and are expressed as: mean ± standard deviation. The data were statistically analyzed using T-test. When P < 0.05, it indicates that there is a significant difference in the embryo incidence rate between different generations of materials, which is marked with *; when P < 0.01, it indicates that there is an extremely significant difference in the embryo incidence rate between different generations of materials, which is marked with **.

[0070] The results in Table 1 show that the preparation method for obtaining high-frequency embryogenesis material of plants provided by the present invention can indeed obtain high-frequency embryogenesis material, and the effect is very obvious for materials with low embryogenesis rate. After one generation of screening, the embryogenesis rate can be increased to about 50%, which can be used for subsequent genetic transformation. For high-frequency embryogenesis materials, the effect of the method is not significant, but after multiple consecutive screenings, the embryogenesis rate of the material can be continuously improved. After field identification, the agronomic traits of the high-frequency embryogenesis material have no obvious changes, such as Figure 6 shown.

[0071] It can be seen from the above experimental results that the method for obtaining high-frequency embryogenesis materials of plants provided by the present invention can effectively screen out individuals with high-frequency embryogenesis ability in crop varieties and retain them, thereby continuously improving the embryogenesis rate in the variety. The method provided by the present invention successfully increased the embryogenesis rate of the more widely used millet varieties Taixuangu 24, Huangmaogu, Jigu 41, and Jigu 39 to more than 40%, so that they can be used for downstream genetic transformation and gene editing, and the millet genetic transformation system is expanded to multiple production varieties. The present invention can solve the problem that the embryogenesis rate of varieties used in production is low and difficult to use for genetic transformation.

[0072] The present invention provides a method for obtaining plant embryogenic materials, which can inoculate mature embryos of low-frequency embryogenic materials on an induction medium to induce the formation of embryonic calli, and induce the formed embryonic calli to regenerate and induce seedlings, and after one or more repeated screenings, high-frequency embryogenic materials under the genotype background can be obtained. The present invention provides a method for obtaining plant embryogenic materials, which does not require overexpression of regeneration genes, uses plant explants as materials, and is induced to culture callus tissue, and then obtains plant high-frequency embryogenic materials through tissue culture screening, and does not introduce exogenous genes. It can not only effectively solve the genotype restriction problem in genetic breeding, but also has the advantages of being relatively simple and easy to operate, not involving complex transgenic operations, not being restricted by relevant laws and regulations, and being able to effectively improve the embryogenesis rate.

[0073] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0074] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A method for obtaining high-frequency embryogenic material of plants, characterized in that: The steps include: inoculating plant explants onto callus induction medium for culture to obtain callus tissue; The callus tissue was subcultured 4 to 6 times continuously; The embryonic callus grown after 4 to 6 subcultures is transferred to a budding medium for culture, and the embryonic callus is induced to form adventitious buds on the budding medium; The adventitious buds are transferred to rooting medium and cultured until roots are formed; The rooted plants are transplanted and cultured until they are mature and strong, thereby obtaining the high-frequency embryogenesis material.

2. The method for obtaining plant high-frequency embryogenic material according to claim 1, characterized in that: The plant explant includes any one of a mature embryo, an immature embryo and a hypocotyl.

3. The method for obtaining plant high-frequency embryogenic material according to claim 1, characterized in that: The conditions for culturing the plant explants on the callus induction medium are: 24° C. to 26° C. and 13 h to 15 h of light.

4. The method for obtaining plant high-frequency embryogenic material according to claim 3, characterized in that: The formula composition of the callus induction medium is: 4.44g / L MS solid powder medium, 30g / L-50g / L sucrose, 0.5mg / L-3mg / L 2,4-D, 0.1mg / L-1mg / L KT, 2.6g / L-4.0g / L plant gel, and the solvent is water.

5. The method for obtaining plant high-frequency embryogenic material according to claim 1, characterized in that: The frequency of subculture is once every 15 to 20 days.

6. The method for obtaining plant high-frequency embryogenic material according to claim 1, characterized in that: The conditions for culturing the embryonic callus on the budding medium are: 24° C. to 26° C. and 13 h to 15 h of light.

7. The method for obtaining plant high-frequency embryogenic material according to claim 6, characterized in that: The budding culture medium comprises: 4.44 g / L MS solid powder culture medium, 30 g / L-50 g / L sucrose, 0.5 mg / L-5 mg / L 6-BA, 2.6 g / L-4.0 g / L plant gel, and the solvent is water.

8. The method for obtaining plant high-frequency embryogenic material according to claim 1, characterized in that: The conditions for culturing the adventitious buds on the rooting medium are: 24° C. to 26° C. and 13 h to 15 h of light per day.

9. The method for obtaining plant high-frequency embryogenic material according to claim 8, characterized in that: The formula of the rooting culture medium is as follows: 2.22 g / L MS solid powder culture medium, 30 g / L-50 g / L sucrose, 0.5 mg / L-5 mg / L IBA, 2.6 g / L-4.0 g / L plant gel, and the solvent is water.

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