Method for improving European spruce somatic embryo development synchronism

Abstract

The invention relates to a method for improving European spruce somatic embryo development synchronism, and relates to the technical field of plant tissue culture. The method comprises the following steps: (1) carrying out induction culture to obtain an embryogenic callus; (2) carrying out subculture multiplication culture on the embryogenic callus to obtain an embryogenic callus cell line; (3) pre-differentiation treatment; (4) differentiation treatment; wherein the differentiation pretreatment liquid and the differentiation culture medium both contain 5-10 [mu] M of EED226. According to the method, a histone methyltransferase inhibitor EED226 is added into a differentiation pretreatment solution and a differentiation culture medium, so that the aims of inhibiting mass proliferation of embryonic calluses in the somatic embryo maturation process, promoting the development and maturation of somatic embryos and improving the development synchronism of the somatic embryos of picea are fulfilled.

Description

Technical Field

[0001] The present application relates to the technical field of plant tissue culture, and in particular to a method for improving the developmental synchronization of somatic embryos of Picea abies. Background Art

[0002] The European spruce (Picea abies (L.) Karst.), also known as Norway spruce and European black pine, is a member of the genus Picea in the Pinaceae family. It is native to northern and central Europe. Since its introduction to my country from Japan in 1926 by the Xiongyue Arboretum, it has been successfully introduced to China in numerous locations (including Jiangxi, Shandong, Liaoning, and Gansu), where it has thrived. It is a successful exotic timber species introduced to northern my country. The European spruce is an evergreen tree with an elegant, upright form, reaching heights of up to 60 meters and a diameter at breast height of 15 meters. It is a fast-growing timber species with high economic value. Its wood, known as white pine, is straight and creamy white, light yellow, or brownish-red, with a fine, uniform structure and excellent strength. It is commonly used in furniture, musical instruments, and interior and exterior wood decoration. It is an important building and structural timber in Europe. Mature European spruce trees have a spire-shaped crown, dense branches, and bright green needles. They are often used as garden trees and are highly ornamental.

[0003] Somatic embryogenesis, the process by which a single cell or tissue undergoes dedifferentiation and redifferentiation in vitro to form a plant, is a crucial technology for coniferous clonal forestry. It enables the efficient and intensive production of large numbers of somatic embryonic seedlings in a short period of time. The resulting seedlings have minimal genetic variation and are uniform. Compared to traditional propagation methods (seed propagation and cuttings), somatic embryogenesis is faster, more scalable, and less costly. Spruce somatic embryogenesis generally proceeds through four stages: induction and acquisition of embryonic callus, maintenance and proliferation of embryonic callus, maturation of somatic embryos, and germination and plantlet regeneration. Plant somatic embryogenesis holds broad potential for application. However, in practice, cell division and differentiation during somatic embryo formation are often asynchronous. Furthermore, the continuous formation of embryonic cells and secondary embryos results in a lack of synchronization in somatic embryo production. Consequently, somatic embryos at different developmental stages can occur within the same culture system, significantly reducing the efficiency of somatic embryo development into complete plants and presenting a significant obstacle to its application. Therefore, improving the synchronization of spruce somatic embryo development is the key to improving production efficiency.

[0004] At present, although somatic embryogenesis systems have been established for many spruce species, most systems have the problem of low synchronization of somatic embryo development, which seriously restricts the application of somatic embryogenesis technology. Summary of the Invention

[0005] In order to solve or partially solve the problems existing in the related art, the present application provides a method for improving the synchronization of somatic embryo development in European spruce. The method adds the histone methyltransferase inhibitor EED226 to the differentiation pretreatment solution and the differentiation culture medium to inhibit the massive proliferation of embryonic callus during the somatic embryo maturation process, promote the development and maturation of somatic embryos, and achieve the purpose of improving the synchronization of somatic embryo development in spruce.

[0006] A method for improving the developmental synchronization of Picea abies somatic embryos in the present application comprises the following steps: (1) Immature zygotic embryos were removed from immature cones of Picea abies, inoculated into induction medium, and induced into embryonic callus; (2) Subculture the embryonic callus to obtain an embryonic callus cell line; (3) Pre-differentiation treatment: Place the embryonic callus in the differentiation pre-treatment solution, spread it out, and culture it in the dark at a temperature of 23±1°C and a shaking speed of 100-120 rpm for 1 week to obtain the embryonic callus after pre-differentiation treatment; The differentiation pretreatment solution formula is: 1 / 2 LM, sucrose 10 g / L, enzymatic hydrolyzed casein 1 g / L, EED226 5-10 μM, pH = 5.8 ± 0.01; (4) Differentiation treatment: Take out the embryonic callus tissue after pre-differentiation treatment, dry the differentiation pre-treatment solution with filter paper, and then place the embryonic callus tissue on differentiation medium for somatic embryo induction culture to induce the formation of somatic embryos; Among them, the culture conditions are: temperature 23±1℃, dark culture for 5-8 weeks; The formula of differentiation medium is: 1 / 2 LM, sucrose 30 g / L, gelatin 4 g / L, enzymatic 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.

[0007] Furthermore, in step (4), the embryonic callus tissue after pretreatment is taken out and the differentiation pretreatment liquid is absorbed by filter paper. The specific operation is as follows: the embryonic callus tissue after pretreatment is taken out and spread on a circular filter paper, and then the circular filter paper is placed on the square filter paper to absorb the differentiation pretreatment liquid.

[0008] Mechanism of this application: Somatic embryogenesis is essentially a process in which somatic cells reshape their developmental fate, regaining cellular totipotency and subsequently differentiating into a complete individual. Epigenetic modifications are upstream of the molecular regulatory network and play a crucial role in reshaping the fate of somatic embryos. Histone modification, as a key mechanism of epigenetics, plays a crucial role in gene regulation and genomic stability. Among them, H3K27me3 modification is a hallmark of gene silencing. By regulating gene expression, it participates in various biological processes, including growth and development, and has a very important biological function. Studies have shown that H3K27me3 levels are correlated with the embryogenic potential of specific tissues, and the early stages of somatic embryogenesis are accompanied by changes in H3K27me3 levels. The primary function of PRC2 is to repress transcription by depositing the repressive histone mark H3K27me3 on target chromatin. Embryonic ectoderm development (EED) is a key member of the PRC2 complex, and its high expression is a key factor in the transition of H3K27me3 modification.

[0009] EED226 is a potent and selective PRC2 inhibitor that directly binds to the H3K27me3 binding pocket of EED. After binding to EED, EED226 causes conformational changes, leading to the loss of PRC2 activity. It can broadly inhibit H3K27me3 modification, thereby affecting the transcriptional repression state of genes, thereby inhibiting the massive proliferation of embryonic tissues during embryonic maturation, promoting the development and maturation of somatic embryos, and achieving the purpose of improving the synchronization of spruce somatic embryo development.

[0010] The beneficial effect of the present application is that the present application can inhibit the massive proliferation of embryonic tissues during embryonic maturation by adding an appropriate amount of histone methyltransferase inhibitor EED226 to the corresponding differentiation pretreatment solution and differentiation culture medium during the differentiation pretreatment stage and the differentiation treatment stage, thereby promoting the development and maturation of somatic embryos and achieving the purpose of improving the synchronization of spruce somatic embryo development. The operation is simple and the synchronization effect of improving the synchronization of somatic embryo development is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The above and other objects, features and advantages of the present application will become more apparent through a more detailed description of exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0012] Figure 1 This is a schematic diagram showing the effects of different concentrations of EED226 on the maturation of somatic embryos of Picea abies. Figure 1Middle AF are pictures of culture dishes of Picea abies differentiation culture for 6 weeks, which are CK, 1 μM, 2 μM, 5 μM, 10 μM, and 0.1% DMSO groups, respectively; Figure 1 Middle GL is a super-depth image (41X) of somatic embryos of Picea abies differentiated for 6 weeks, including CK, 1 μM, 2 μM, 5 μM, 10 μM, and 0.1% DMSO groups. DETAILED DESCRIPTION

[0013] The embodiments of the present application will be described in more detail below with reference to the examples. Although the embodiments of the present application are shown in the examples, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art. Example

[0014] 1. Callus Tissue Source and Cell Line Construction From mid-to-late July 2021 to 2023, immature cones of Picea abies were harvested in Xiaolongshan, Gansu. Cones were disinfected by soaking in 95% alcohol for 15 minutes and then rinsing three times with sterile water for 2 minutes each. The seeds were then removed from the axial surface of the seed scales using scissors and tweezers and placed in a 50 ml conical flask.

[0015] Seeds were sterilized by 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. The seeds were placed on sterile filter paper, and the inner and outer seed coats were removed using a scalpel and forceps. The embryo and endosperm were then placed on induction medium and incubated in the dark at 23 ± 1°C for 6-7 weeks.

[0016] After 6-7 weeks of induction of zygotic embryos on induction medium, callus tissue is isolated and proliferated using the same medium used for induction. The resulting callus tissue is divided into two categories: embryonic callus and non-embryonic callus. Non-embryonic callus is white or light yellow in color and has a loose, granular or compact structure. Embryonic callus is white, translucent, and has filamentous projections. Microscopically, an embryonic head with a large nucleus and dense cytoplasm and a cluster of filamentous suspensor cells can be observed.

[0017] During subculture, 7-9 pieces of embryonic callus with a diameter of 0.5 cm were propagated in one culture dish (90 mm). The cells were subcultured once every 2 weeks and cultured in the dark at 23±1°C. A cell line of a certain scale could be formed after 2 months.

[0018] The starting materials used in the following examples were immature cones of Picea abies collected randomly from Xiaolongshan, Gansu Province, between 2021 and mid-July 2023. These cones were labeled and used as embryonic callus induced from immature zygotic embryos. Embryonic calli were numbered according to the year of collection and the order of observation, such as 21Pa-1, which represents the first callus cell line discovered from cones collected in 2021.

[0019] 2. Culture medium configuration The culture medium was prepared according to the following formula: 1 / 2 LM medium was used as the base (as shown in Table 1), and different concentrations of hormones such as 2,4-D (2,4-dichlorophenoxyacetic acid), 6-BA (6-benzylaminopurine), and ABA (abscisic acid) were added, as well as sucrose, plant gelatin, and enzymatically hydrolyzed casein.

[0020] Induction medium formula: 1 / 2 LM, 2,4-D (2.2 mg / L), 6-BA (1.1 mg / L), sucrose (10 g / L), gelatin (4 g / L), enzymatic hydrolyzed casein (1 g / L), (pH = 5.8 ± 0.01).

[0021] Proliferation medium formula: same as induction medium.

[0022] Differentiation basic pretreatment solution: 1 / 2 LM, sucrose (10 g / L), enzymatic hydrolyzed casein (1 g / L), (pH = 5.8 ± 0.01).

[0023] Differentiation basic medium: 1 / 2 LM, sucrose (30 g / L), gelatin (4 g / L), enzymatic hydrolyzed casein (1 g / L), ABA (24 mg / L), PEG4000 (50 g / L), activated carbon (1 g / L), (pH = 5.8 ± 0.01).

[0024] Table 1 1 / 2LM basic culture medium formula

[0025] EED226 stock solution configuration: Weigh 10 mg of EED226 powder and dissolve it in 2.707 ml of DMSO to prepare a 10 mM stock solution. Aliquot the stock solution and store at -80°C to avoid repeated freeze-thaw cycles.

[0026] 3. Synchronous Developmental Regulation The synchronization control method was to add different concentrations of EED226 to the differentiation basic pretreatment solution and differentiation basic medium, respectively, with no EED226 and 0.1% DMSO added as controls. The specific experimental groups are as follows (Table 2): Table 2 Overall plan for grouping in the synchronization control experiment

[0027] 1. Pre-differentiation treatment Specifically, during the pre-differentiation treatment, fresh (5-7 days old) embryogenic callus cells were used as the differentiation material. 2.5 g of embryogenic callus cell line (line number 22Pa-5) was weighed and placed in 150 ml of basic differentiation pre-treatment solution. EED226 was then added to the basic differentiation pre-treatment solution and basic differentiation medium to form the corresponding pre-differentiation solution and differentiation medium. Finally, the cells were incubated in a constant temperature shaker at 110 rpm, 23 ± 1°C, and in the dark for one week. Simultaneously, control groups were established, with experiments numbered 1 and 6 serving as the control group. The experimental protocol is shown in Table 3.

[0028] Table 3 Pretreatment solution for differentiation of embryogenic callus cell lines of Picea abies

[0029] 2. Differentiation Using a pipette, 3 ml of the differentiation pretreatment solution from experimental groups 1-6, obtained after one week of dark culture, was evenly spread onto a 60 mm circular filter paper. The circular filter paper was then placed on a 15 cm square filter paper to drain the differentiation pretreatment solution. The circular filter paper was then placed in differentiation medium containing varying concentrations of EED226 (the drug concentration in the differentiation medium was consistent with that in the differentiation pretreatment solution). The culture dishes were incubated in the dark at 23 ± 1°C for 6 weeks, and the somatic embryo synchronization rate was calculated. The results are shown in Table 4. When 1 or 2 μM EED226 was added to the differentiation pretreatment solution and differentiation medium, the somatic embryo synchronization rate was not significantly different from that of the control group (experimental group 1) without EED226. When 5 μM EED226 was added to the differentiation pretreatment solution and differentiation medium, the somatic embryo synchronization rate was 26.74%, a 39.23% increase compared to experimental group 1. When 10 μM EED226 was added to the differentiation basic pretreatment solution and differentiation basic culture medium, the somatic embryo synchronization rate was 47.79%, which was an increase of 150.08% compared with experimental group 1.

[0030] That is to say, when 5-10 μM EED226 is added to both the differentiation basic pretreatment solution and the differentiation basic culture medium, the synchronization of somatic embryo development of embryonic callus tissue will be significantly improved.

[0031] Mature somatic embryo synchronization rate (%) = number of mature somatic embryos / total number of embryos × 100% Table 4 Effects of different concentrations of EED226 on somatic embryo maturation of Picea abies

[0032] in, Figure 1 This is a schematic diagram showing the effects of different concentrations of EED226 on the maturation of somatic embryos of Picea abies. Figure 1 Middle AF are pictures of culture dishes of Picea abies differentiation culture for 6 weeks, which are CK, 1 μM, 2 μM, 5 μM, 10 μM, and 0.1% DMSO groups, respectively; Figure 1 Middle GL is a super-depth image (41X) of somatic embryos of Picea abies differentiated for 6 weeks, including CK, 1 μM, 2 μM, 5 μM, 10 μM, and 0.1% DMSO groups.

[0033] from Figure 1 It can be clearly seen that compared with the control group CK (A, G) and 0.1% DMSO (F, L), 5-10 μM EED226 treatment (DE, JK) can significantly increase the somatic embryo synchronization rate.

[0034] 3. Effects of adding EED226 at different stages on the synchronization of European spruce Based on the above basic operations of pre-differentiation and differentiation treatment, the effects of adding EED226 at different stages on the synchronization of European spruce were investigated. The experimental scheme is shown in Table 5, and the experimental results are shown in Table 6: Table 5 Effects of adding EED226 at different stages on the synchronization of European spruce

[0035] Note: “+” indicates the addition of 10 μM EED226, “-” indicates no treatment, and cells 7 and 8 are control groups.

[0036] Table 6 Effects of different treatment groups at the same concentration on somatic embryo maturation of Picea abies

[0037] As can be seen from Table 6, the treatments in Experimental Groups 3 and 5 were both effective in improving the synchronization rate. However, since EED226 was added to the proliferation, pre-differentiation, and differentiation stages in Experimental Group 3, considering the principle of reducing the number of operation boxes and saving reagents, it is preferred to add EED226 only in the pre-differentiation and differentiation stages.

[0038] IV. Changes in proliferation weight during the entire culture process Table 7 shows that throughout somatic embryo development, tissues undergo significant proliferation after one week of differentiation pretreatment and one week of differentiation, then slow down until tissue weight stabilizes at four weeks of differentiation. Furthermore, the addition of 10 μM EED226 to the basic differentiation pretreatment solution and basic differentiation medium significantly inhibits embryonic tissue proliferation. After five weeks of differentiation, tissue weight was reduced by 51.06% compared to treatment group 1.

[0039] Table 7 Effects of different concentrations of EED226 on the proliferation of embryonic tissue

[0040] The embodiments of the present application have been described above. The above description is illustrative and not exhaustive, and is not 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 selected to best explain the principles of the embodiments, their practical applications, or improvements to the technology in the market, or to enable other persons skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for improving the developmental synchronization of somatic embryos of Picea abies, characterized in that: The following steps are involved: (1) Immature zygotic embryos were removed from immature cones of Picea abies, inoculated into induction medium, and induced into embryonic callus; (2) Subculture the embryonic callus to obtain an embryonic callus cell line; (3) Pre-differentiation treatment: The embryonic callus cell line was placed in a differentiation pre-treatment solution. After spreading, the cells were cultured in the dark at a temperature of 23 ± 1 °C and a shaking speed of 100-120 rpm for 1 week to obtain the embryonic callus after pre-differentiation treatment. The differentiation pretreatment solution formula is: 1 / 2 LM, sucrose 10 g / L, enzymatic hydrolyzed casein 1 g / L, EED2265-10 μM, pH = 5.8 ± 0.01; (4) Differentiation treatment: Take out the embryonic callus tissue after pre-differentiation treatment, dry the differentiation pre-treatment solution with filter paper, and then place the embryonic callus tissue on differentiation medium for somatic embryo induction culture to induce the formation of somatic embryos; Among them, the culture conditions are: temperature 23±1℃, dark culture for 5-8 weeks; The formula of differentiation medium is: 1 / 2 LM, sucrose 30 g / L, gelatin 4 g / L, enzymatic 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. A method for improving the developmental synchronization of Picea abies somatic embryos according to claim 1, characterized in that: In step (4), the embryonic callus tissue after pretreatment is taken out and the differentiation pretreatment liquid is absorbed by filter paper. The specific operation is as follows: the embryonic callus tissue after pretreatment is taken out and spread on a circular filter paper, and then the circular filter paper is placed on a square filter paper to absorb the differentiation pretreatment liquid.

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