A method for improving the synchronization of somatic embryo development in Picea abies

Abstract

This application relates to a method for improving the synchronicity of somatic embryo development in European spruce, and pertains to the field of plant tissue culture technology. The method includes the following steps: (1) inducing and culturing into embryogenic callus; (2) subculturing the embryogenic callus to obtain an embryogenic callus cell line; (3) predifferentiation treatment; (4) differentiation treatment; wherein the predifferentiation treatment solution and the differentiation medium both contain 5-10 μM of EED226. This method, by adding the histone methyltransferase inhibitor EED226 to the predifferentiation treatment solution and the differentiation medium, aims to inhibit the excessive proliferation of embryogenic callus during somatic embryo maturation, promote the development and maturation of somatic embryos, and thus improve the synchronicity of somatic embryo development in spruce.

Description

Technical Field

[0001] This application relates to the field of plant tissue culture technology, and in particular to a method for improving the synchronization of somatic embryo development in European spruce. Background Technology

[0002] European spruce (Picea abies (L.) Karst.), also known as Norway spruce or European black spruce, is a plant belonging to the genus *Picea* in the family Pinaceae. Native to Northern and Central Europe, it was introduced to my country from Japan in 1926 by the Xiong Yue Arboretum. It has been successfully introduced and tested in various regions (Jiangxi, Shandong, Liaoning, Gansu, etc.) and has shown good growth, making it a successful introduced timber species in northern my country. European spruce is an evergreen tree with a beautiful, straight shape, reaching up to 60 meters in height and 15 meters in diameter at breast height. It is a fast-growing timber species with high economic value. Its wood, known as white pine, is straight, creamy white, pale yellow, or brownish-red in color, with a fine and uniform texture and excellent strength. It is a common material for furniture, musical instruments, and other decorative applications, primarily used in interior and exterior woodwork. It is an important building and structural material in Europe. Mature European spruce trees have pyramidal crowns, dense branches, and bright green needles, making them a popular garden tree species with high ornamental value.

[0003] Somatic embryogenesis refers to the process by which a single cell or tissue dedifferentiates and redifferentiates to form a plant under in vitro conditions. It is an important technical means for achieving clonal coniferous forestry, enabling the efficient and intensive production of large quantities of somatic embryonic seedlings in a short period of time. The seedlings produced have small differences in genetic background and are uniform in size. Compared with traditional propagation methods (seed propagation and cutting propagation), it is faster, larger in scale, and lower in cost. Spruce somatic embryogenesis generally goes through four stages: induction and acquisition of embryogenic callus, maintenance and proliferation of embryogenic callus, maturation of somatic embryos, and germination and regeneration of somatic embryos. Plant somatic embryogenesis has broad application prospects. However, in practice, the asynchronous cell division and differentiation during somatic embryo formation, coupled with the continuous formation of embryogenic cells and secondary embryos, leads to low synchronicity in somatic embryo production. That is, somatic embryos at different developmental stages exist in the same culture system, which seriously reduces the efficiency of somatic embryos developing into complete plants, becoming the biggest obstacle to the application of this system. Therefore, improving the synchronicity of spruce embryo development is key to improving production efficiency.

[0004] Currently, although somatic embryogenesis systems have been established for various spruce species, most systems suffer from low synchronicity in somatic embryo development, which severely restricts the application of somatic embryogenesis technology. Summary of the Invention

[0005] To address or partially address the problems existing in the related technologies, this application provides a method for improving the synchronicity of somatic embryo development in European spruce. This method involves adding the histone methyltransferase inhibitor EED226 to the differentiation pretreatment solution and differentiation culture medium to inhibit the excessive proliferation of embryogenic callus during somatic embryo maturation, promote the development and maturation of somatic embryos, and thereby improve the synchronicity of somatic embryo development in spruce.

[0006] A method for improving the synchronization of somatic embryonic development in European spruce trees, as described in this application, includes the following steps:

[0007] (1) Immature zygotic embryos were taken from immature cones of European spruce and inoculated into induction medium for induction culture to form embryogenic callus;

[0008] (2) The embryonic callus tissue was subcultured and proliferated to obtain an embryonic callus cell line;

[0009] (3) Predifferentiation treatment: The embryogenic callus was placed in the differentiation pretreatment solution, and after spreading out, it was cultured in the dark for 1 week at a temperature of 23±1℃ and a shaking speed of 100-120 rpm to obtain the predifferentiated embryogenic callus.

[0010] The differentiation pretreatment solution is formulated as follows: 1 / 2 LM, 10 g / L sucrose, 1 g / L enzymatically hydrolyzed casein, 5-10 μM EED226, pH=5.8 ± 0.01;

[0011] (4) Differentiation treatment: Take out the predifferentiated embryonic callus, blot dry the differentiation pretreatment solution with filter paper, and then place the embryonic callus on the differentiation medium for somatic embryo induction culture to induce the formation of somatic embryos;

[0012] The cultivation conditions were: temperature 23±1℃, dark culture for 5-8 weeks;

[0013] The differentiation medium was formulated as follows: 1 / 2 LM, sucrose 30 g / L, gel 4 g / L, enzymatically hydrolyzed casein 1 g / L, ABA 24 mg / L, PEG4000 50 g / L, activated carbon 1 g / L, EED226 5-10 μM, pH=5.8 ± 0.01.

[0014] Further, in step (4), the pretreated embryogenic callus is removed and the differentiation pretreatment solution is absorbed by filter paper. Specifically, the pretreated embryogenic callus is taken out and spread flat on a circular filter paper, and then the circular filter paper is placed on a square filter paper to absorb the differentiation pretreatment solution.

[0015] The mechanism of this application: Somatic embryogenesis is essentially a process in which the developmental fate of somatic cells is reshaped, regaining totipotency and thus redifferentiating into a complete individual. Epigenetic modifications, located upstream in the molecular regulatory network, play a crucial role in the reshaping of somatic embryonic fate. Histone modifications, as an important mechanism in epigenetics, play a vital role in gene regulation and genome stability. Among them, H3K27me3 modification is a marker of gene silencing, participating in various biological processes such as growth and development by regulating gene expression, and has very important biological functions. Studies have shown that H3K27me3 levels are related to the embryogenetic potential of specific tissues, and changes in H3K27me3 levels accompany the early stages of somatic embryogenesis. The main function of PRC2 is to inhibit transcription by depositing the repressive histone marker H3K27me3 on target chromatin. Embryonic ectoderm development (EED) is an important member of the PRC2 complex, and its high expression is an important reason for the over-modification of H3K27me3.

[0016] EED226 is an effective and selective PRC2 inhibitor that binds directly to the H3K27me3 binding pocket of EED. After binding to EED, EED226 causes a conformational change, resulting in the loss of PRC2 activity. It can broadly inhibit the modification of H3K27me3, thereby affecting the transcriptional repression state of genes. This inhibits the excessive proliferation of embryonic tissues during embryonic maturation, promotes the development and maturation of somatic embryos, and achieves the goal of improving the synchronicity of spruce somatic embryonic development.

[0017] The beneficial effects of this application are: by adding an appropriate amount of histone methyltransferase inhibitor EED226 to the differentiation pretreatment solution and differentiation culture medium during the differentiation pretreatment and differentiation treatment stages, this application can inhibit the excessive proliferation of embryonic tissues during embryo maturation, promote the development and maturation of somatic embryos, and achieve the purpose of improving the synchronicity of somatic embryo development in spruce. The operation is simple and the synchronization effect of improving somatic embryo development is good. Attached Figure Description

[0018] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments thereof.

[0019] Figure 1 This is a schematic diagram illustrating the effect of different concentrations of EED226 treatment on the embryo maturation of European spruce.

[0020] Figure 1The images in the middle (AF) show culture dishes of European spruce after 6 weeks of differentiation culture, representing the CK, 1 μM, 2 μM, 5 μM, 10 μM, and 0.1% DMSO groups, respectively.

[0021] Figure 1 The image in GL is a super depth-of-field image (41X) of somatic embryos cultured for 6 weeks after differentiation of European spruce, representing the CK, 1 μM, 2 μM, 5 μM, 10 μM, and 0.1% DMSO groups, respectively. Detailed Implementation

[0022] The embodiments of this application will now be described in more detail with reference to the examples. While embodiments of this application are shown in the examples, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art. Example

[0023] I. Callus origin and cell line construction

[0024] From mid to late July 2021 to 2023, immature cones of European spruce were harvested in Xiaolongshan, Gansu Province. The cones were disinfected by soaking in 95% alcohol for 15 minutes and rinsing three times with sterile water for two minutes each time. Using scissors and tweezers, the seeds were separated from the seed scales facing the axis and placed in 50 ml Erlenmeyer flasks.

[0025] Seed disinfection was performed as follows: soaking in 75% alcohol for 1 minute, rinsing twice with sterile water, soaking in 2% sodium hypochlorite solution for 10 minutes, and rinsing five times with sterile water. Seeds were then placed on sterile filter paper, and the inner and outer seed coats were peeled off using a scalpel and forceps. The embryo and endosperm were placed on induction medium and cultured in the dark at 23±1℃ for 6-7 weeks.

[0026] After 6-7 weeks of induction on the zygote culture medium, the callus tissue was isolated and proliferated using the same culture medium as the induction medium. The induced callus tissue was divided into two types: embryogenic callus and non-embryonic callus. Non-embryonic callus was white or pale yellow in color, and either loosely granular or densely structured. Embryogenic callus appeared white, crystalline, translucent, and had filamentous protrusions. Under a microscope, large-nucleated embryonic heads with dense cytoplasm and clusters of fine-filamentous embryonic stalk cells could be observed.

[0027] During subculture, 7-9 embryogenic callus tissues with a diameter of 0.5 cm are proliferated in one culture dish (90 mm). Subculture is performed once every 2 weeks. The cells are cultured in the dark at 23±1℃. After 2 months, a cell line of a certain size can be formed.

[0028] The original materials used in the following case studies were immature cones of European spruce randomly collected from Xiaolongshan, Gansu Province, between 2021 and mid-July 2023. These cones were labeled and used as starting material for embryogenic callus induced from immature zygotes. The embryogenic callus was numbered according to the year of collection and the order of observation; for example, 21Pa-1 represents the first callus cell line discovered after induction from cones collected in 2021.

[0029] II. Culture medium preparation

[0030] The culture medium was prepared according to the following formula: based on 1 / 2 LM medium (as shown in Table 1), different concentrations of hormones such as 2,4-D (2,4-dichlorophenoxyacetic acid), 6-BA (6-benzylaminopurine), and ABA (abscisic acid) were added, along with sucrose, plant gel, and enzymatically hydrolyzed casein.

[0031] Induction medium formulation: 1 / 2 LM, 2,4-D (2.2 mg / L), 6-BA (1.1 mg / L), sucrose (10 g / L), gel (4 g / L), enzymatically hydrolyzed casein (1 g / L), (pH=5.8 ± 0.01).

[0032] The proliferation medium formula is the same as the induction medium.

[0033] Differentiation pretreatment solution: 1 / 2 LM, sucrose (10 g / L), enzymatically hydrolyzed casein (1 g / L), (pH=5.8 ±0.01).

[0034] Differentiation basal medium: 1 / 2 LM, sucrose (30 g / L), gel (4 g / L), enzymatically hydrolyzed casein (1 g / L), ABA (24 mg / L), PEG4000 (50 g / L), activated carbon (1 g / L), (pH=5.8 ± 0.01).

[0035] Table 1 1 / 2LM Basic Culture Medium Formulation

[0036]

[0037] EED226 stock solution preparation:

[0038] Weigh 10 mg of EED226 powder and dissolve it in 2.707 ml of DMSO solution to prepare a 10 mM stock solution. Aliquot the stock solution and store it in a freezer at -80°C, avoiding repeated freeze-thaw cycles.

[0039] III. Synchronous Development Regulation

[0040] The synchronization control method involved adding different concentrations of EED226 to the differentiation pretreatment solution and differentiation basal medium, with no EED226 and 0.1% DMSO serving as controls. The specific experimental groups are as follows (Table 2):

[0041] Table 2 Overall Scheme for Synchronization Control Experiment Groups

[0042]

[0043] 1. Pre-differentiation treatment

[0044] Specifically, fresh (5-7 days old) embryogenic callus cell lines were used as differentiation material during predifferentiation treatment. 2.5 g of the embryogenic callus cell line (line number 22Pa-5) was weighed and placed in 150 ml of basic differentiation pretreatment solution. The tissue was dispersed, and different concentration gradients of EED226 were added to the basic differentiation pretreatment solution and basic differentiation medium to prepare the corresponding differentiation pretreatment solution and differentiation medium. Finally, the mixture was placed in a constant temperature shaker at 110 rp, 23±1℃, and incubated in the dark for 1 week. Meanwhile, control groups were set up, with experiments numbered 1 and 6 serving as control groups. The specific experimental protocol is shown in Table 3.

[0045] Table 3. Pretreatment solution for differentiation of embryogenic callus cell lines from European spruce.

[0046]

[0047] 2. Differentiation treatment

[0048] Using a pipette, 3 ml of the differentiation pretreatment solution from experimental groups 1-6, after one week of dark incubation, was evenly spread on a 60 mm circular filter paper. The circular filter paper was then placed on a 15 cm square filter paper to absorb the pretreatment solution. The circular filter paper was then placed in differentiation medium containing different concentrations of EED226 (the drug concentration in the differentiation medium was the same as that in the differentiation pretreatment solution). The culture dishes were placed in a constant temperature incubator at 23±1℃ and incubated in the dark for 6 weeks. The somatic embryo synchronization rate was recorded, and the results are shown in Table 4. It can be seen that when 1 or 2 μM of EED226 was added to the basic differentiation pretreatment solution and basic differentiation medium, the somatic embryo synchronization rate was not significantly different from the control group (experimental group 1) without EED226. When 5 μM of EED226 was added to the basic differentiation pretreatment solution and basic differentiation medium, the somatic embryo synchronization rate was 26.74%, which was 39.23% higher than that of experimental group 1. When 10 μM of EED226 was added to the differentiation pretreatment solution and differentiation basal medium, the somatic embryo synchronization rate was 47.79%, which was 150.08% higher than that of experimental group 1.

[0049] In other words, adding 5-10 μM of EED226 to both the basic differentiation pretreatment solution and the basic differentiation medium can significantly improve the somatic embryonic development synchronization of embryogenic callus.

[0050] Mature embryo synchronization rate (%) = Number of mature embryos / Total number of embryos × 100%

[0051] Table 4. Effects of different concentrations of EED226 on embryo maturation of European spruce.

[0052]

[0053] in, Figure 1 This is a schematic diagram illustrating the effect of different concentrations of EED226 treatment on the embryo maturation of European spruce.

[0054] Figure 1 The images in the middle (AF) show culture dishes of European spruce after 6 weeks of differentiation culture, representing the CK, 1 μM, 2 μM, 5 μM, 10 μM, and 0.1% DMSO groups, respectively.

[0055] Figure 1 The image in GL is a super depth-of-field image (41X) of somatic embryos cultured for 6 weeks after differentiation of European spruce, representing the CK, 1 μM, 2 μM, 5 μM, 10 μM, and 0.1% DMSO groups, respectively.

[0056] from Figure 1 It is evident that, compared to the control groups CK (A, G) and 0.1% DMSO (F, L), treatment with 5-10 μMEED226 (DE, JK) significantly improves the somatic embryo synchronization rate.

[0057] 3. The effect of EED226 treatment at different stages on the synchronicity of European spruce

[0058] Based on the basic operations of the pre-differentiation and differentiation treatments described above, the effects of adding EED226 at different stages on the synchronicity of European spruce were investigated. The experimental protocol is shown in Table 5, and the experimental results are shown in Table 6.

[0059] Table 5. Effects of EED226 treatment at different stages on the synchronicity of European spruce.

[0060]

[0061] Note: "+" indicates the addition of 10 μM EED226, "-" indicates no treatment, and 7 and 8 are control groups.

[0062] Table 6. Effects of different treatment groups at the same concentration on embryo maturation of European spruce.

[0063]

[0064] As shown in Table 6, the treatments in experimental groups 3 and 5 can effectively improve the synchronization rate. However, since EED226 was added in experimental group 3 during the proliferation, pre-differentiation, and differentiation stages, considering the principle of reducing the number of operation boxes and saving reagents, it is preferable to add EED226 only during the pre-differentiation and differentiation stages.

[0065] IV. Changes in proliferation weight throughout the entire culture process

[0066] Table 7 shows that during the entire somatic embryonic development process, tissues undergoing differentiation pretreatment and differentiation for one week experience significant proliferation, followed by a slowdown in proliferation rate, with tissue weight gradually stabilizing by week 4 of differentiation. Furthermore, the addition of 10 μM EED226 to the basic differentiation pretreatment solution and basic differentiation medium significantly inhibited the proliferation of embryogenic tissues; after week 5 of differentiation, the tissue weight was reduced by 51.06% compared to treatment group 1.

[0067] Table 7. Effects of different concentrations of EED226 on embryogenic tissue proliferation.

[0068]

[0069] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for improving the synchronization of somatic embryonic development in European spruce, characterized in that, Includes the following steps: (1) Immature zygotic embryos were taken from immature cones of European spruce and inoculated into induction medium for induction culture to form embryogenic callus; (2) The embryonic callus tissue was subcultured and proliferated to obtain an embryonic callus cell line; (3) Predifferentiation treatment: The embryonic callus cell line was placed in the differentiation pretreatment solution, and after spreading it out, it was cultured in the dark for 1 week at a temperature of 23±1℃ and a shaking speed of 100-120 rpm to obtain the predifferentiated embryonic callus. The differentiation pretreatment solution is formulated as follows: 1 / 2 LM, 10 g / L sucrose, 1 g / L enzymatically hydrolyzed casein, EED 2265-10 μM, pH=5.8 ± 0.01; (4) Differentiation treatment: Take out the predifferentiated embryonic callus, blot dry the differentiation pretreatment solution with filter paper, and then place the embryonic callus on the differentiation medium for somatic embryo induction culture to induce the formation of somatic embryos; The cultivation conditions were: temperature 23±1℃, dark culture for 5-8 weeks; The differentiation medium was formulated as follows: 1 / 2 LM, sucrose 30 g / L, gel 4 g / L, enzymatically hydrolyzed casein 1 g / L, ABA 24 mg / L, PEG4000 50 g / L, activated carbon 1 g / L, EED226 5-10 μM, pH=5.8 ± 0.

01.

2. The method for improving the synchronization of somatic embryonic development in European spruce according to claim 1, characterized in that, In step (4), the pretreated embryogenic callus is removed and the differentiation pretreatment solution is absorbed with filter paper. Specifically, the pretreated embryogenic callus is taken out and spread flat on a circular filter paper. Then, the circular filter paper is placed on a square filter paper to absorb the differentiation pretreatment solution.

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