A method for obtaining high frequency embryogenic material of plants
By screening through tissue culture and acclimatizing to specific culture media, high-frequency embryogenesis materials were obtained, which solved the genotype limitation in crop variety genetic transformation, improved the embryogenesis rate, simplified the operation process, and is applicable to many varieties that are widely promoted in production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANXI AGRI UNIV
- Filing Date
- 2024-12-06
- Publication Date
- 2026-04-28
AI Technical Summary
In existing technologies, many crop varieties are difficult to genetically transform, mainly due to the lack of regenerative genes. Explant-induced callus tissue cannot undergo embryogenesis or has a low embryogenesis rate, which limits gene editing and transgenic breeding.
Through tissue culture screening, specific culture media and conditions are used for acclimatization and screening to obtain high-frequency embryogenetic materials, including callus subculture, embryogenic callus transfer and rooting culture, avoiding the introduction of exogenous genes and simplifying the operation process.
It increases the embryogenesis rate of crop varieties, solves the problem of genotype restriction, simplifies the operation process, avoids complicated genetic modification operations and legal restrictions, and is suitable for varieties that are widely promoted in production.
Smart Images
Figure CN119605646B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant tissue culture and genetic transformation technology, specifically relating to a method for obtaining high-frequency embryogenesis materials in plants. Background Technology
[0002] Currently, genetic transformation systems have been established for various crops, enabling gene editing breeding. However, due to genotype limitations, only a few varieties / lines of these crops can be used for genetic transformation, while varieties widely used in production are often difficult to directly transform. Genotype limitations in the genetic transformation process are prevalent in various crops, significantly restricting gene editing and transgenic breeding, and becoming one of the biggest bottlenecks in these fields. This is mainly because most breeding materials lack regenerable genes; the callus induced by their explants cannot undergo embryogenesis or has a very low embryogenesis rate, resulting in a low regeneration rate after tissue culture, failure to produce seedlings, and inability to obtain successfully transformed seeds. To widely apply genetic transformation or gene editing breeding technologies in crop breeding, the genotype limitation problem in crop genetic breeding must be solved.
[0003] Currently, overexpressing embryonic development genes through transgenic technology can effectively solve the genotype restriction problem in crop genetic breeding. However, this method introduces exogenous genes, involves complex transgenic operations, and is therefore difficult to implement. Summary of the Invention
[0004] To address the problem that existing genetic transformation technologies, such as overexpression of embryonic development genes via transgenic technology, introduce foreign genes, involve complex transgenic operations, and are difficult to implement, this invention provides a method for obtaining high-frequency embryogenesis materials from plants.
[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.
[0006] This invention provides a method for obtaining high-frequency embryogenesis material in plants, comprising the following steps:
[0007] The plant explants were disinfected, cleaned, and then inoculated onto callus induction medium to obtain callus tissue. The callus tissue was subcultured 4 to 6 times, with subculture every 15 to 20 days.
[0008] Embryogenic callus tissues that have grown after 4 to 6 subcultures are transferred to budding medium for further culture, where adventitious buds are induced from the embryogenic callus tissues. The formation of embryogenic callus tissues is a crucial step in tissue culture regeneration. Selected embryogenic callus tissues are transferred to budding medium, while callus tissues that cannot form embryogenic callus tissues are discarded.
[0009] Adventitious buds were transferred to rooting medium and cultured until they rooted.
[0010] After the plants have taken root, they are transplanted and cultivated until they mature and bear fruit to obtain the high-frequency embryogenesis material.
[0011] This invention provides a method for obtaining plant embryogenetic material. Mature embryos of low-frequency embryogenetic material are inoculated onto an induction medium to induce the formation of embryogenic callus. The resulting callus is then regenerated and induced into seedlings. Through one or more repeated screenings, high-frequency embryogenetic material with the desired genotype can be obtained. This method does not require overexpression of regeneration genes. Using plant explants as material, callus tissue is induced and cultured, and then high-frequency embryogenetic material suitable for plant genetic transformation is obtained through continuous tissue culture screening. This method not only effectively solves the genotype limitation problem in genetic breeding but also has the advantages of being relatively simple and easy to implement, not introducing exogenous genes, not involving complex transgenic operations, not being restricted by relevant laws and regulations, and effectively improving the embryogenesis rate.
[0012] Meanwhile, the method for obtaining plant embryogenesis materials provided by this 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.
[0013] Preferably, the plant explant comprises any one of a mature embryo, an immature embryo, and a hypocotyl. These materials induce callus regeneration with strong regenerative capacity and are well-suited for embryogenic callus formation.
[0014] Preferably, the plant explants are cultured on the callus induction medium under the following conditions: 24℃~26℃, 13h~15h light exposure. More preferably, the plant explants are cultured on the callus induction medium under the following conditions: 25℃, 14h light exposure.
[0015] Preferably, the callus induction medium is formulated as follows: MS solid powder medium 4.44 g / L, sucrose 30 g / L to 50 g / L, 2,4-D 0.5 mg / L to 3 mg / L, KT 0.1 mg / L to 1 mg / L, plant gel 2.6 g / L to 4.0 g / L, and water as the solvent. This medium formulation can effectively induce the formation of embryogenic callus from plant material.
[0016] Because the embryogenesis rates of widely used varieties in current production are low, they are difficult to use for genetic transformation. Therefore, the high-frequency embryogenesis materials currently suitable for plant genetic transformation are primarily not those widely used in production. For example, Hi-II and B104, commonly used for genetic transformation of maize, and Ci846, used for genetic transformation of millet, are not varieties widely used in current production. Establishing a genetic transformation system for widely used varieties requires optimizing the conditions of the entire plant tissue culture, which is often unsuccessful. Moreover, the optimal culture conditions identified through this method cannot be replicated in other varieties.
[0017] The method for obtaining plant embryogenesis material provided by this invention improves the embryogenesis rate of a variety by selecting individuals suitable for that culture medium through domestication and screening on the same medium, followed by selective purification. This avoids the need for extensive optimization of the culture medium. Furthermore, the method successfully increases the embryogenesis rate of widely used millet varieties such as Taixuangu 24, Huangmaogu, Jigu 41, and Jigu 39 to over 40%, enabling downstream genetic transformation and gene editing work and expanding the millet genetic transformation system to multiple production varieties.
[0018] Preferably, the subculture frequency is once every 15 days.
[0019] Preferably, the embryogenic callus is cultured on the budding medium under the following conditions: 24℃~26℃, 13h~15h light exposure. More preferably, the embryogenic callus is cultured on the budding medium under the following conditions: 25℃, 14h light exposure.
[0020] Preferably, the budding medium is formulated as follows: MS solid powder medium 4.44 g / L, sucrose 30 g / L to 50 g / L, 6-BA 0.5 mg / L to 5 mg / L, plant gel 2.6 g / L to 4.0 g / L, and water as the solvent. This medium can effectively induce the formation of adventitious buds from embryogenic callus in plants.
[0021] Preferably, the adventitious buds are cultured on the rooting medium under the following conditions: 24℃~26℃, with 13h~15h light per day. More preferably, the adventitious buds are cultured on the rooting medium under the following conditions: 25℃, with 14h light per day.
[0022] Preferably, the rooting medium is formulated as follows: MS solid powder medium 2.22 g / L, sucrose 30 g / L to 50 g / L, IBA 0.5 mg / L to 5 mg / L, plant gel 2.6 g / L to 4.0 g / L, and water as the solvent. This medium can induce adventitious buds of plants to rapidly develop into well-developed root systems.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] 1. This invention provides a method for obtaining high-frequency embryogenetic material in plants. The method provides for obtaining high-frequency embryogenetic material in plants through tissue culture screening, without introducing exogenous genes. This not only effectively solves the genotype limitation 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 effectively improving the embryogenesis rate.
[0025] 2. The method for obtaining high-frequency embryogenesis material in plants provided by this invention, through domestication and screening on the same culture medium, selects individuals suitable for that culture medium from a certain variety, and then purifies them, thereby improving the embryogenesis rate of that variety and avoiding extensive optimization of the culture medium. Simultaneously, the method for obtaining high-frequency embryogenesis material in plants provided by this invention can effectively screen out individuals with high-frequency embryogenesis ability in crop varieties and retain them, thereby continuously improving the embryogenesis rate of that variety.
[0026] 3. The method for obtaining high-frequency embryogenesis materials provided by the present invention involves screening out individuals that cannot develop embryos from the selected variety / line population through tissue culture, while retaining individuals with embryogenesis ability. After one or more rounds of tissue culture, the frequency of genes with high-frequency embryogenesis rate in low-frequency embryogenesis materials can be increased, thereby increasing the embryogenesis rate of the population and achieving the need for genetic transformation.
[0027] 4. This invention allows for the propagation or rooting of adventitious buds formed after tissue culture-induced embryogenic callus, resulting in high-frequency embryogenetic materials that are not significantly different from the original variety.
[0028] 5. Although overexpressing embryonic development genes through transgenic technology can effectively solve the genotype restriction problem in genetic breeding, the overexpression of these embryonic development genes often results in unfavorable phenotypes, leading to abnormal plant development, which must be removed through backcrossing after transformation. Therefore, compared with the method of obtaining high-frequency embryogenetic material of the present invention, the method of overexpressing embryonic development genes through transgenic technology leads to a longer breeding cycle and more cumbersome operation. The method of obtaining high-frequency embryogenetic material of the present invention does not rely on the plant's genetic transformation system, does not introduce exogenous genes, and can improve low-frequency embryogenetic material.
[0029] 6. The improved material of this invention can stably maintain the characteristic of high embryogenesis rate, and can be used to solve the problem of low embryogenesis rate and difficulty in genetic transformation of varieties that are produced and promoted. Attached Figure Description
[0030] Figure 1 This refers to the case in Example 1 of this invention where mature embryo seeds were inoculated onto callus induction medium for 15 days after inoculation.
[0031] Figure 2 This refers to the embryogenic callus formed on the callus induction culture medium in Example 1 of the present invention.
[0032] Figure 3 The term refers to the clustered shoots formed by embryogenic callus on the budding culture medium in Example 1 of this invention.
[0033] Figure 4 This is the case of inducing rooting from clustered buds in Example 1 of the present invention.
[0034] Figure 5 This refers to the case where R1 generation seeds from Example 1 of the present invention are inoculated on callus induction medium.
[0035] Figure 6 The field performance of the high-frequency embryogenesis materials in Examples 1 and 5 of this invention is shown; wherein, Figure 6 In this context, A represents Jin Gu 51; Figure 6 B in the text refers to Jin Valley 51R1; Figure 6 C in the text is Jigu 39; Figure 6 D in the equation is Jigu 39R1. Detailed Implementation
[0036] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.
[0037] Example 1
[0038] The plant material used in this embodiment is millet.
[0039] A method for obtaining high-frequency embryogenesis material in plants includes the following steps:
[0040] (1) Explant disinfection: Millet seeds were used as explants, and the generation of the material used was named R0. They were disinfected with 75% alcohol for 1 min, then with 10% sodium hypochlorite solution for 20 min. Finally, the millet seeds were washed 5 times with sterile water, placed on sterile filter paper to absorb excess moisture on the seed surface, and inoculated onto callus induction medium to induce callus tissue.
[0041] Among them, the millet seeds are of the variety Jin Gu 51, which comes from the College of Agriculture of Shanxi Agricultural University.
[0042] The callus induction medium was formulated as follows: 4.44 g / L MS solid powder medium, 30 g / L sucrose, 2 mg / L 2,4-D, 0.5 mg / L KT, and 2.6 g / L plant gel, with water as the solvent, pH 5.8, and sterilized at 121℃ for 20 min. The MS solid powder medium was purchased from Phytotech.
[0043] (2) Callus induction: The callus induction medium inoculated with explants was placed in a tissue culture room at 25°C under 14-hour light. Subculture was performed approximately every 15 days, transferring the resulting callus to fresh callus induction medium. Browned or water-soaked callus was discarded. Figure 1 As shown.
[0044] (3) Embryogenic callus formation: After three subcultures, granular embryogenic callus forms successively. The formed embryogenic callus is as follows: Figure 2 As shown, the formation of embryogenic callus is a key step in tissue culture regeneration. Selected embryogenic callus is transferred to budding medium, while callus that cannot form embryogenic callus is discarded.
[0045] The germination medium was formulated as follows: 4.44 g / L MS solid powder medium, 30 g / L sucrose, 0.5 mg / L 6-BA, and 2.6 g / L plant gel, with water as the solvent, pH 5.8, and sterilized at 121°C for 20 min. The MS solid powder medium was purchased from Phytotech.
[0046] (4) Sprouting: After transferring the embryogenic callus to a sprouting medium, adventitious buds emerge from the embryogenic callus in about 20 days. The formed adventitious buds are as follows: Figure 3 As shown. This adventitious bud can be further propagated on the budding medium for the next experiment. Once the adventitious bud reaches 2cm in length, it is transferred to the rooting medium. After approximately 25 days, slender hairy roots will develop, forming roots as shown... Figure 4 As shown.
[0047] The rooting medium consisted of: 2.22 g / L MS solid powder medium, 30 g / L sucrose, 0.5 mg / L IBA, and 2.6 g / L plant gel, with water as the solvent, pH 5.8, and sterilized at 121°C for 20 min. The MS solid powder medium was purchased from Phytotech.
[0048] (5) Transplanting and harvesting: Transplant the seedlings with hairy roots into a moist seedling substrate, cover them with plastic wrap to keep them moist, and after the seedlings gradually adapt, remove the plastic wrap, water them regularly, maintain a temperature of about 25℃ and 14h / 10h light. After the seedlings flower and bear fruit, harvest the seeds, which are named R1 generation.
[0049] The seedling substrate is made by mixing peat moss and vermiculite in a 7:3 ratio.
[0050] It should be noted that the R1 generation seeds are the high-frequency embryogenesis materials obtained.
[0051] (6) Determination of embryogenesis rate in harvested seeds: The harvested R1 generation seeds were inoculated onto callus induction medium using the methods described in steps (1)-(3) to induce embryogenic callus tissue. The embryogenesis rate of the R1 generation seeds after tissue culture selection was improved, such as... Figure 5 As 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 the R2 generation seeds. At this time, the R2 generation seeds are the high-frequency embryogenesis material obtained.
[0053] Example 2
[0054] A method for obtaining high-frequency embryogenesis material in plants includes the following steps:
[0055] Similar to Example 1, except that the millet seeds were of the Taixuangu 24 variety and were sourced from the College of Agriculture, Shanxi Agricultural University.
[0056] Example 3
[0057] A method for obtaining high-frequency embryogenesis material in plants includes the following steps:
[0058] Similar to Example 1, except that the variety of millet seeds is yellow-haired millet, and the source is the Millet Research Institute of Shanxi Agricultural University.
[0059] Example 4
[0060] A method for obtaining high-frequency embryogenesis material from plants includes the following steps:
[0061] Similar to Example 1, except that the millet seeds were of variety Jigu 41 and were sourced from the College of Agriculture, Shanxi Agricultural University.
[0062] Example 5
[0063] A method for obtaining high-frequency embryogenesis material in plants includes the following steps:
[0064] Similar to Example 1, except that the variety of millet seeds was Jigu 39, which came from the College of Agriculture of Shanxi Agricultural University.
[0065] To illustrate the effectiveness of the above method, the embryogenesis rates of R0, R1, and R2 generation seeds in Examples 1 to 5 were statistically analyzed. The specific methods are as follows:
[0066] R0, R1, and R2 generation seeds were inoculated onto callus induction medium, and the embryogenesis rate at 60 days was recorded. The results are shown in Table 1.
[0067] Table 1. Embryogenesis at 160 days
[0068] Variety Name R0 generation% R1 generation% R2 generation% Jingu 51 62.95±2.93 63.23±6.42 75.48±3.70* Taisuke Valley 24 32.44±6.05 54.98±1.00* / Yellow Hair Valley 9.50±2.10 55.08±6.21** / Jigu 41 4.52±2.27 41.22±2.07** / Jigu 39 9.13±1.10 59.21±3.00** /
[0069] Note: Data in the table are repeated three times and are expressed as mean ± standard deviation. The data were analyzed using a T-test. When P < 0.05, it indicates a significant difference in embryogenesis rate among different generations of materials, marked with *; when P < 0.01, it indicates a highly significant difference in embryogenesis rate among different generations of materials, marked with **.
[0070] The results in Table 1 show that the method for preparing high-frequency embryogenesis materials provided by this invention can indeed obtain materials with high embryogenesis rates. The effect is very significant for materials with low embryogenesis rates, increasing the embryogenesis rate to approximately 50% after one generation of screening, which can then be used for subsequent genetic transformation. For high-frequency embryogenesis materials, the improvement effect of this method is not significant, but after multiple consecutive screenings, the embryogenesis rate of the materials can be continuously improved. Furthermore, field evaluation showed no significant changes in the agronomic traits of the high-frequency embryogenesis materials. Figure 6 As shown.
[0071] The experimental results above demonstrate that the method for obtaining high-frequency embryogenesis materials in plants provided by this invention can effectively screen and retain individuals with high-frequency embryogenesis ability in crop varieties, thereby continuously improving the embryogenesis rate in these varieties. The method provided by this invention has successfully increased the embryogenesis rate of commonly used millet varieties such as Taixuangu 24, Huangmaogu, Jigu 41, and Jigu 39 to over 40%, enabling its application in downstream genetic transformation and gene editing, and expanding the millet genetic transformation system to multiple production varieties. This invention solves the problem of low embryogenesis rates in production varieties, making them difficult to use for genetic transformation.
[0072] This invention provides a method for obtaining plant embryogenetic material. Mature embryos of low-frequency embryogenetic material are inoculated onto an induction medium to induce the formation of embryogenic callus. The resulting embryogenic callus is then regenerated and induced into seedlings. After one or more repeated screenings, high-frequency embryogenetic material with the specified genotype can be obtained. This method does not require overexpression of regeneration genes. Using plant explants as material, it induces callus formation and then obtains high-frequency embryogenetic material through tissue culture screening. Without introducing exogenous genes, it effectively solves the genotype limitation problem in genetic breeding, is relatively simple and easy to implement, does not involve complex transgenic operations, is not subject to relevant laws and regulations, and effectively improves the embryogenesis rate.
[0073] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.
[0074] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.
Claims
1. A method for obtaining high-frequency embryogenesis material in plants, characterized in that, Includes the following steps: Plant explants were inoculated onto a callus induction medium to obtain callus tissue. The callus induction medium consisted of: 4.44 g / L MS solid powder medium, 30 g / L to 50 g / L sucrose, 0.5 mg / L to 3 mg / L 2,4-D, 0.1 mg / L to 1 mg / L KT, and 2.6 g / L to 4.0 g / L plant gel, with water as the solvent. The callus tissue was continuously subcultured 4 to 6 times; Embryogenic callus that has grown after 4 to 6 subcultures is transferred to a sprouting medium for culture, where adventitious shoots are induced from the embryogenic callus. The sprouting medium is formulated as follows: MS solid powder medium 4.44 g / L, sucrose 30 g / L to 50 g / L, 6-BA 0.5 mg / L to 5 mg / L, plant gel 2.6 g / L to 4.0 g / L, with water as the solvent. Adventitious buds were transferred to rooting medium and cultured until rooting occurred; the rooting medium consisted of: MS solid powder medium 2.22 g / L, sucrose 30 g / L to 50 g / L, IBA 0.5 mg / L to 5 mg / L, plant gel 2.6 g / L to 4.0 g / L, and water as the solvent. After rooting, the plants are transplanted and cultured until they mature and bear fruit. R1 generation seeds are then harvested to obtain the high-frequency embryogenesis material. The harvested R1 generation seeds are inoculated onto a callus induction medium to induce embryogenic callus. The embryogenesis rate of the R1 generation seeds is improved after tissue culture selection. If the embryogenesis rate of the R1 generation seeds does not meet the application requirements, the above steps are repeated to obtain R2 generation seeds. The R2 generation seeds are the obtained high-frequency embryogenesis material. The plant is millet ( Setaria italica ).
2. The method for obtaining high-frequency embryogenesis material in plants according to claim 1, characterized in that, The plant explants include any one of mature embryos, immature embryos, and hypocotyls.
3. The method for obtaining high-frequency embryogenesis material in plants according to claim 1, characterized in that, The plant explants were cultured on the callus induction medium under the following conditions: 24℃~26℃, 13h~15h light exposure.
4. The method for obtaining high-frequency embryogenesis material in plants according to claim 1, characterized in that, The subculture frequency is once every 15 to 20 days.
5. The method for obtaining high-frequency embryogenesis material in plants according to claim 1, characterized in that, The embryogenic callus was cultured on the budding medium under the following conditions: 24℃~26℃, 13h~15h light exposure.
6. The method for obtaining high-frequency embryogenesis material in plants according to claim 1, characterized in that, The conditions for culturing the adventitious buds on the rooting medium are: 24℃~26℃, 13h~15h light per day.