Application of DNA methylation inhibitor in promoting somatic embryogenesis of taxodium 'zhongshannan'

Abstract

The application belongs to the technical field of plant somatic embryogenesis, and particularly relates to application of a DNA methylation inhibitor in promoting somatic embryogenesis of Taxodium 'Zhongshanshan', wherein the DNA methylation inhibitor comprises 5-AzaC. The application adds 5-AzaC in a somatic embryo induction medium and a somatic embryo germination and seedling raising medium, so that somatic embryo induction efficiency of Taxodium 'Zhongshanshan' is improved, induction speed is accelerated, somatic embryo induction rate of low-embryo genotypes is realized from low to high, and somatic embryo seedling raising rate is greatly improved. Meanwhile, the operation steps of the scheme are simple, only a lower concentration of 5-AzaC needs to be added in the somatic embryo induction medium or the somatic embryo germination and seedling raising medium, so that the somatic embryo induction rate is improved, the somatic embryogenesis process is accelerated, and the seedling raising rate can reach more than 95%. In addition, the method of the application does not need drying treatment, and the seedling raising time is greatly shortened. The application has important significance for breeding and popularization and application of Taxodium 'Zhongshanshan'.

Description

Technical Field

[0001] This invention belongs to the field of plant somatic embryogenesis technology, specifically relating to the application of DNA methylation inhibitors in promoting somatic embryogenesis of Taxus chinensis. Background Technology

[0002] Metasequoia glyptostroboides is a series of superior varieties bred through interspecific hybridization in the genus Taxus. It possesses advantages such as rapid growth, tolerance to salinity and alkalinity, and tolerance to waterlogging. It is widely used in the construction of ecological barriers in low-lying wetlands of the Yangtze River basin and surrounding lakes, as well as saline-alkali lands along the southeast coast of my country, generating significant ecological, economic, and social benefits and generating substantial social demand. Traditionally, the main method for propagating superior Metasequoia glyptostroboides clones is through softwood cuttings. However, during the cutting propagation process, it has been found that as the physiological age of the Metasequoia glyptostroboides clones increases and multiple generations of cutting propagation occur, the rooting rate of the cuttings gradually decreases. Furthermore, there are significant differences in rooting ability and rooting quality among different varieties. These problems severely limit the propagation and widespread application of superior Metasequoia glyptostroboides varieties.

[0003] Somatic embryogenesis refers to the process by which somatic cells are induced to form azygotic embryos under certain conditions, which then germinate into complete plants. This technique has advantages such as high propagation volume and speed. However, the current somatic embryogenesis system of Metasequoia glyptostroboides still has problems such as low somatic embryogenesis efficiency and large differences in somatic embryogenesis frequency among different varieties, which require further optimization of the Metasequoia glyptostroboides somatic embryogenesis system. Summary of the Invention

[0004] The purpose of this invention is to provide the application of DNA methylation inhibitors in promoting somatic embryogenesis in Taxus chinensis. This invention involves adding a low concentration of 5-AzaC to the somatic embryo induction or somatic embryo germination seedling culture medium, which can increase the somatic embryo induction rate, accelerate the somatic embryogenesis process, and achieve a seedling rate of over 95%.

[0005] This invention provides the application of DNA methylation inhibitors in promoting somatic embryogenesis of Taxus chinensis, wherein the DNA methylation inhibitors include 5-AzaC.

[0006] The present invention also provides a method for promoting somatic embryogenesis of Taxus chinensis, comprising the following steps:

[0007] The callus tissue of Metasequoia glyptostroboides was initially cultured in a somatic embryo induction medium to obtain cultured Metasequoia glyptostroboides callus tissue.

[0008] The cultured Metasequoia callus was subjected to somatic embryo induction culture in a somatic embryo induction medium containing DNA methylation inhibitors to obtain mature Metasequoia somatic embryos.

[0009] The mature somatic embryos of *Taxus chinensis* were cultured in a somatic embryo germination and seedling culture medium containing DNA methylation inhibitors to obtain *Taxus chinensis* somatic embryo seedlings.

[0010] As a preferred embodiment, the somatic embryo induction medium uses DCR as the basal medium and further includes the following components at concentrations: glutamine 0.3–0.5 g·L⁻¹ -1 CH 0.2~0.5g·L -1 Inositol 0.1–1 g·L -1 PEG 170~210g·L -1 ABA 7~9mg·L -1 GA 3-5 mg / L -1 Maltose 25-35 g / L -1 Aspartic acid 0.1–0.3 g·L -1 Proline 0.1–0.3 g·L -1 Activated carbon 2-3 g / L -1 and plant gel 3-4 g·L -1 .

[0011] As a preferred embodiment, the initial culture of somatic embryo induction is a dark culture, the temperature of the initial culture of somatic embryo induction is 21-25℃, and the time of the initial culture of somatic embryo induction is 2 weeks.

[0012] As a preferred embodiment, the DNA methylation inhibitor is 5-AzaC, and the concentration of 5-AzaC during the somatic embryo induction culture is 5–15 μmol·L⁻¹. -1 .

[0013] As a preferred embodiment, the somatic embryo induction culture is a dark culture, the temperature of the somatic embryo induction culture is 21-25℃, and the time of the somatic embryo induction culture is 6-7 weeks.

[0014] As a preferred embodiment, the somatic embryo germination and seedling culture medium uses WPM as the basal medium and further includes the following components at concentrations: sucrose 20–30 g·L⁻¹ -1 7-8 g / L of agar -1 .

[0015] As a preferred embodiment, the concentration of 5-AzaC during the somatic embryo germination and seedling culture is 5–15 μmol·L⁻¹. -1 .

[0016] As a preferred embodiment, the photoperiod for somatic embryo germination and seedling culture is 16 h / d, and the photoperiod intensity is 230–290 μmol·m⁻¹. -2 .s -1 The temperature for the somatic embryo germination and seedling culture is 21–25℃.

[0017] As a preferred embodiment, the Metasequoia callus includes T170 genotype callus and T3 genotype callus.

[0018] Beneficial Effects: This invention provides the application of DNA methylation inhibitors in promoting somatic embryogenesis in *Taxus chinensis*, wherein the DNA methylation inhibitor includes 5-AzaC. By adding 5-AzaC to the somatic embryo induction medium and the somatic embryo germination and seedling formation medium, this invention improves the efficiency and speed of somatic embryo induction in *Taxus chinensis*, achieving a high somatic embryo induction rate for low-embryo genotypes and significantly increasing the somatic embryo seedling formation rate. Furthermore, the method of this invention is simple to operate; only a low concentration of 5-AzaC needs to be added to the somatic embryo induction medium or the somatic embryo germination and seedling formation medium to increase the somatic embryo induction rate, accelerate the somatic embryogenesis process, and achieve a seedling formation rate of over 95%. In addition, the method of this invention does not require drying treatment, greatly shortening the seedling formation time. This is of great significance for the breeding and promotion of superior *Taxus chinensis* varieties. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 The images show a comparison of the microstructure of the callus tissue in Example 1, where the concentrations of 5-AzaC in a–f and (a)–(f) are 0 μmol·L⁻¹ from left to right. -1 5 μmol·L -1 10 μmol·L -1 15 μmol·L -1 30 μmol·L -1 and 50 μmol·L -1 In the figures, a to f are comparison images of the whole dish, and (a) to (f) are comparison images of the microstructure of the callus tissue.

[0021] Figure 2 The effect of different concentrations of 5-AzaC on callus proliferation in Example 1;

[0022] Figure 3 The effect of different concentrations of 5-AzaC on the number of mature embryos in Example 3;

[0023] Figure 4 To illustrate the effect of different concentrations of 5-AzaC and different induction periods on somatic embryo induction in Example 4, the concentrations of 5-AzaC from left to right are 5 μmol·L⁻¹. -1 10 μmol·L -1 15 μmol·L -1 30 μmol·L -1or 50 μmol·L -1 ;

[0024] Figure 5 The effect of different concentrations of 5-AzaC on the germination and seedling formation of mature embryos is shown in Example 5. From left to right, the concentrations of 5-AzaC are 0 μmol·L⁻¹. -1 5 μmol·L -1 10 μmol·L -1 and 15 μmol·L -1 ;

[0025] Figure 6 The effect of 5-AzaC on the induction of T3 genotype callus somatic embryos in Example 6. Detailed Implementation

[0026] This invention provides the application of DNA methylation inhibitors in promoting somatic embryogenesis of Taxus chinensis, wherein the DNA methylation inhibitors include 5-AzaC.

[0027] The present invention also provides a method for promoting somatic embryogenesis of Taxus chinensis, comprising the following steps:

[0028] The callus tissue of Metasequoia glyptostroboides was initially cultured in a somatic embryo induction medium to obtain cultured Metasequoia glyptostroboides callus tissue.

[0029] The cultured Metasequoia callus was subjected to somatic embryo induction culture in a somatic embryo induction medium containing DNA methylation inhibitors to obtain mature Metasequoia somatic embryos.

[0030] The mature somatic embryos of *Taxus chinensis* were cultured in a somatic embryo germination and seedling culture medium containing DNA methylation inhibitors to obtain *Taxus chinensis* somatic embryo seedlings.

[0031] As a specific implementation method, the Metasequoia glyptostroboides callus can be selected from Metasequoia glyptostroboides callus subcultured for 10 days, and the subculture medium can be a callus proliferation blank medium: DCR as the basal medium, supplemented with 0.45 g·L⁻¹ glutamine. -1 CH (hydrolyzed casein) 0.5 g·L -1 Inositol 0.1 g·L -1 2 mg / L NAA (naphthaleneacetic acid) -1 Maltose 20g·L -1 Activated carbon 2.5 g·L -1 Plant-based gel 2.8 g·L -1 Adjust the pH to 5.8.

[0032] The somatic embryo induction medium of this invention uses DCR as the basic medium and also includes the following components at concentrations: glutamine 0.3-0.5 g·L⁻¹-1 CH (hydrolyzed casein) 0.2-0.5 g·L -1 Inositol 0.1–1 g·L -1 PEG (polyethylene glycol) 170-210 g·L -1 ABA (abscisic acid) 7-9 mg / L -1 GA (gibberellin) 3-5 mg / L -1 Maltose 25-35 g / L -1 Aspartic acid 0.1–0.3 g·L -1 Proline 0.1–0.3 g·L -1 Activated carbon 2-3 g / L -1 and plant gel 3-4 g·L -1 In one specific embodiment, the somatic embryo induction medium uses DCR as the basal medium and further includes the following components at the following concentrations: glutamine 0.45 g·L⁻¹ -1 CH 0.5g·L -1 Inositol 0.1 g·L -1 PEG 190g·L -1 ABA 8mg·L -1 GA4mg·L -1 30g / L maltose -1 Aspartic acid 0.2 g·L -1 Proline 0.2 g·L -1 Activated carbon 2g·L -1 Plant-based gel 3g·L -1 The pH was adjusted to 6.0; as another specific embodiment, the somatic embryo induction medium used DCR as the basal medium and also included the following components at the following concentrations: glutamine 0.3 g·L⁻¹ -1 CH 0.2g·L -1 Inositol 0.1 g·L -1 PEG 170g·L -1 ABA 7mg·L -1 GA3 mg·L -1 25g / L maltose -1 Aspartic acid 0.1 g·L -1 Proline 0.1 g·L -1 Activated carbon 2g·L -1 Plant-based gel 3g·L -1 The pH was adjusted to 6.0; as another specific embodiment, the somatic embryo induction medium used DCR as the basal medium and also included the following components at the following concentrations: glutamine 0.5 g·L⁻¹ -1 CH 0.5g·L -1 Inositol 1g·L -1PEG 210g·L -1 ABA 9mg·L -1 GA 5mg·L -1 35g / L maltose -1 Aspartic acid 0.3 g·L -1 Proline 0.3 g·L -1 Activated carbon 3g·L -1 Plant-based gel 4g·L -1 Adjust the pH to 6.0.

[0033] The somatic embryo induction initial culture of this invention is a dark culture, the temperature of the somatic embryo induction initial culture is 21-25℃, and the culture time is 2 weeks. As a specific embodiment, the temperature of the somatic embryo induction initial culture can be 21℃, 22℃, 23℃, 24℃, or 25℃. The embodiments of this invention show that when the somatic embryo induction initial culture time is 2 weeks, the somatic embryo maturation effect after 5-AzaC treatment is better than when the somatic embryo induction initial culture time is 1 week or 3 weeks. When the somatic embryo induction initial culture time is 2 weeks, the somatic embryos are milky white, uniformly distributed, and dense.

[0034] The DNA methylation inhibitor described in this invention is 5-AzaC, and the concentration of 5-AzaC during the somatic embryo induction culture is 5–15 μmol·L⁻¹. -1 In one specific implementation, the concentration of 5-AzaC can be 5 μmol·L⁻¹. -1 6 μmol·L -1 7 μmol·L -1 8 μmol·L -1 9 μmol·L -1 10 μmol·L -1 11 μmol·L -1 12 μmol·L -1 13 μmol·L -1 14 μmol·L -1 or 15 μmol·L -1 In this invention, 5-AzaC (5-azacytidine) acts as a DNA methylation inhibitor, suppressing DNA methyltransferase activity and reducing the overall methylation level of the genome. The role of 5-AzaC in this invention may be to break the silencing state of certain genes (such as key genes in embryonic development) through demethylation, activate the expression of related genes, and further improve the somatic embryo induction rate or synchronization degree.

[0035] The somatic embryo induction culture described in this invention is a dark culture, the temperature of the somatic embryo induction culture is 21–25°C, and the culture time is 6–7 weeks. As a specific embodiment, the temperature of the somatic embryo induction culture can be 21°C, 22°C, 23°C, 24°C, or 25°C. As a specific embodiment, the culture time can be 6 weeks or 7 weeks.

[0036] The somatic embryo germination and seedling culture medium of the present invention uses WPM as the basic medium and also includes the following components at the following concentrations: sucrose 20-30 g·L⁻¹ -1 7-8 g / L of agar -1 In one specific implementation, the somatic embryo germination and seedling culture medium uses WPM as the basic medium and also includes the following components at the following concentrations: sucrose 30 g·L⁻¹ -1 and 8g·L of agar -1 The pH was adjusted to 5.8; as another specific implementation, the somatic embryo germination and seedling culture medium used WPM as the basic medium and also included the following components at the following concentrations: sucrose 20 g·L⁻¹ -1 and 7g·L of agar -1 The pH was adjusted to 5.8; as another specific implementation, the somatic embryo germination and seedling culture medium used WPM as the basic medium and also included the following components at the following concentrations: sucrose 25 g·L⁻¹ -1 and 7.3 g·L agar -1 The pH was adjusted to 5.8; as another specific implementation, the somatic embryo germination and seedling culture medium used WPM as the basic medium and also included the following components at the following concentrations: sucrose 28 g·L⁻¹ -1 and 7.5 g·L agar -1 Adjust the pH to 5.8.

[0037] The concentration of 5-AzaC during the somatic embryo germination and seedling culture described in this invention is 5–15 μmol·L⁻¹. -1 In one specific implementation, the concentration of 5-AzaC can be 5 μmol·L⁻¹. -1 6 μmol·L -1 7 μmol·L -1 8 μmol·L -1 9 μmol·L -1 10 μmol·L -1 11 μmol·L -1 12 μmol·L -1 13 μmol·L -1 14 μmol·L -1 or 15 μmol·L -1 As a specific implementation method, the concentration of 5-AzaC in the somatic embryo germination to seedling culture is 10 μmol·L⁻¹.-1 At this time, the seedling survival rate is the highest, reaching 95%.

[0038] The photoperiod for somatic embryo germination and seedling culture described in this invention is 16 h / d, and the photoperiod intensity is 230–290 μmol·m⁻¹. -2 .s -1 The temperature for the somatic embryo germination and seedling culture is 21–25°C. As a specific embodiment, the light intensity can be 230 μmol·m⁻². -2 .s -1 240 μmol.m -2 .s -1 250 μmol·m -2 .s -1 260 μmol.m -2 .s -1 270 μmol·m -2 .s -1 280 μmol·m -2 .s -1 Or 290 μmol.m -2 .s -1 In one specific implementation, the temperature for the somatic embryo germination and seedling culture can be 21℃, 22℃, 23℃, 24℃, or 25℃.

[0039] The callus tissue of *Taxus chinensis* described in this invention includes T170 genotype callus tissue and T3 genotype callus tissue. As a specific embodiment, the T3 genotype callus tissue is a low-embryonic genotype callus tissue, meaning that this genotype has an extremely low embryogenicity. This invention utilizes 10 μmol·L⁻¹ of methyl methacrylate (MTBF) added to the somatic embryo induction medium. -1 The 5-AzaC molecule enabled the low embryogenic T3 genotype callus to achieve a high somatic embryo induction rate, resulting in the differentiation of more cotyledonary embryos from the low embryogenic callus.

[0040] This invention adds 5-AzaC to the somatic embryo induction medium and the somatic embryo germination and seedling formation medium, thereby improving the somatic embryo induction efficiency and accelerating the induction rate of Metasequoia glyptostroboides. It achieves a significant increase in somatic embryo induction rate for low-embryo genotypes and greatly improves the seedling formation rate. Furthermore, the method is simple to operate; adding only a low concentration of 5-AzaC to the somatic embryo induction or germination and seedling formation medium is sufficient to increase the somatic embryo induction rate, accelerate the somatic embryogenesis process, and achieve a seedling formation rate of over 95%. In addition, the method eliminates the need for drying treatment, greatly shortening the seedling formation time. This invention is of great significance for the breeding and widespread application of superior Metasequoia glyptostroboides varieties.

[0041] To further illustrate the present invention, the application of the DNA methylation inhibitor provided by the present invention in promoting somatic embryogenesis of Taxodium distichum is described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0042] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0043] Example 1

[0044] T170 genotype callus (Chen T, Wang D, Yuan G, et al. High-and low-embryogenic Taxodium hybrid 'zhongshanshan' cell lines reveal involvement of redox homeostasis in somatic embryogenesis[J]. Scientia Horticulturae, 2025, 339. DOI:10.1016 / j.scienta.2024.113830.) was used as the culture material.

[0045] Effect of 5-AzaC on callus induction: Callus proliferation blank medium (using DCR (Coollabo; catalog number: PM1631) as the basal medium, supplemented with 0.45 g·L⁻¹ glutamine) -1 CH 0.5g·L -1 Inositol 0.1 g·L -1 NAA2 mg·L -1 Maltose 20g·L -1 Activated carbon 2.5 g·L -1 Gel 2.8g·L -1 After adjusting the pH of the culture medium to 5.8, it was sterilized at 121°C for 20 min. Once the culture medium cooled to 60°C, filtered sterile 5-AzaC (final concentration 0 μmol·L⁻¹) was added. -1 5 μmol·L -1 10 μmol·L -1 15 μmol·L -1 30 μmol·L -1 and 50 μmol·L -1 After 10 days of subculture, T170 genotype embryogenic callus was selected from vigorous callus that was in a transparent state and inoculated into a solution containing 0 μmol·L⁻¹. -1 5 μmol·L -1 10 μmol·L -115 μmol·L -1 30 μmol·L -1 and 50 μmol·L -1 The cells were cultured in subculture medium containing 5-azacytidine (5-AzaC, Shanghai Aladdin, catalog number A100625) (i.e., blank medium for callus proliferation containing different concentrations of 5-AzaC). All cultures were incubated in the dark at 23°C. After 15 days of culture, the fresh weight of the embryogenic callus was weighed. A small sample of the callus was then placed on a glass slide, distilled water was added, and the sample was gently dispersed with tweezers. A coverslip was then placed on the slide, and the cell morphology was observed under a microscope.

[0046] In this experiment, at least three replicate culture dishes were set up for each treatment, and six pieces of callus tissue, approximately 0.2g each, were inoculated into each dish. The initial fresh weight of the callus tissue at inoculation was recorded as W0, and the fresh weight of the callus tissue after culture was recorded as W1. The formula for calculating the fresh weight of callus proliferation was W1-W0, and the proliferation rate was calculated using the formula (W1-W0) / W0×100%.

[0047] Results analysis:

[0048] The effects of different concentrations of 5-AzaC on the proliferation of T170 genotype callus are shown in the figure. Figure 1 (The concentration from left to right is 0 μmol·L⁻¹) -1 5 μmol·L -1 10 μmol·L -1 15 μmol·L -1 30 μmol·L -1 and 50 μmol·L -1 The top image shows a comparison of whole-plate images, and the bottom image shows a comparison of the microstructure of callus tissue. The results show that, in the study of embryogenic callus proliferation, 5-AzaC treatment significantly inhibited its proliferation effect compared to the control group. Control group embryogenic callus ( Figure 1 The volume of a) was significantly larger than that of each 5-AzaC treatment group ( Figure 1 (middle bf). Morphological assessment showed that the control group ( Figure 1 a) and 5-AzaC treatment group ( Figure 1 Both bf and fen exhibit a similar white and transparent appearance, with a moist surface and no significant structural differences. Figure 1 (a)-(f)).

[0049] Statistical analysis revealed that ( Figure 2 As the concentration of 5-AzaC increased from the control group (0 μmol·L⁻¹), -1 Increased to 50 μmol·L -1The proliferation rate of embryogenic callus decreased significantly from approximately 350% initially to around 30% (P<0.05). This indicates that the addition of 5-AzaC significantly inhibited the proliferation of embryogenic callus.

[0050] Example 2

[0051] T170 genotype callus tissue that had been subcultured for 10 days was selected as the culture material. Somatic embryo induction medium was based on DCR (Coollab; catalog number: PM1631) supplemented with 0.45 g·L⁻¹ glutamine. -1 CH (hydrolyzed casein) 0.5 g·L -1 Inositol 0.1 g·L -1 PEG (polyethylene glycol) 190g·L -1 (Shanghai Yuanye Biotechnology, Product No. V32174), ABA (abscisic acid) 8mg·L -1 GA (gibberellin) 4 mg·L -1 (Shanghai Yuanye Biotechnology, Product No. S28506), Maltose 30g·L -1 Aspartic acid 0.2 g·L -1 Proline 0.2 g·L -1 Activated carbon 2g·L -1 Plant-based gel 3g·L -1 The pH was adjusted to 6.0. The final concentration of 5-AzaC was set to 0 μmol·L⁻¹. -1 5 μmol·L -1 10 μmol·L -1 and 15 μmol·L -1 Somatic embryo induction medium was added to each embryo. After 60 days of culture, the occurrence of somatic embryos was recorded, and the number of mature embryos obtained by induction was calculated. Somatic embryo induction was performed in the dark at a temperature of 23℃.

[0052] The results are shown in Table 1. The study found that low-concentration 5-AzaC treatment accelerated somatic embryogenesis, while high-concentration 5-AzaC treatment inhibited its efficiency. Mature cotyledonary embryos appeared in the CK treatment at week 6. (5-AzaC 5 μmol·L⁻¹) -1 and 10 μmol·L -1 Mature cotyledonous embryos appeared in week 5 of the treatment, one week earlier than the control group.

[0053] Table 1. Time of emergence of mature cotyledon embryos after treatment with different concentrations of 5-AzaC.

[0054]

[0055] Example 3

[0056] In the somatic embryo induction experiment, T170 genotype callus subcultured for 10 days was selected as the culture material. The callus subcultured for 10 days was then inoculated with a solution of 0 μmol·L⁻¹. -1 5 μmol·L -1 10 μmol·L -1 15 μmol·L -1 30 μmol·L -1 and 50 μmol·L -1 5-AzaC somatic embryo induction medium. The medium was based on DCR basal medium, supplemented with hydrolyzed casein (0.5 g·L⁻¹). -1 Inositol (0.1 g·L) -1 ), polyethylene glycol (190 g·L) -1 (Shanghai Yuanye Biotechnology, catalog number V32174), glutamine (0.45 g·L) -1 ), aspartic acid (0.2 g·L) -1 ), proline (0.2 g·L) -1 ), maltose (30g·L) -1 ), activated carbon (2 g·L) -1 ), plant gel (3g·L) -1 Abscisic acid (ABA, 8 mg·L) -1 ) and gibberellin (GA, 4 mg·L) -1 (Shanghai Yuanye Biotechnology, catalog number S28506). After adjusting the pH of the culture medium to 6.0, all other components were autoclaved at 121℃ for 20 min. ABA, GA, and 5-AzaC, which had undergone aseptic filtration, were added after the culture medium cooled to 60℃. All cultures were incubated in the dark at 23℃, and the tissue structure was observed under a microscope every 5 days. Each treatment had at least three replicates, with each dish inoculated with six embryogenic callus pieces (approximately 0.2 g). After 60 days of culture, the number of columnar embryos (N1) and mature embryos (N2) was counted, and the somatic embryo maturity rate was calculated using the formula: N2 / (N1+N2)×100%.

[0057] Results analysis:

[0058] From the perspective of somatic embryo induction efficiency ( Figure 3 ), 5 μmol·L -1 10 μmol·L -1 and 15 μmol·L -1 The number of mature cotyledon embryos increased with 5-AzaC treatment at 10 μmol·L⁻¹. -1 The treatment yielded the highest maturity rate, with approximately nine mature embryos per dish. (30 μmol·L⁻¹) -1 and 50 μmol·L -15-AzaC treatment resulted in slower SE development and a lower maturity rate compared to the control, with an average of approximately 5 and 3 mature embryos per dish.

[0059] The above results indicate that an appropriate concentration of 5-AzaC can accelerate the development of somatic embryos and increase the somatic embryo maturity rate.

[0060] Example 4

[0061] T170 genotype callus tissue that had been subcultured for 10 days was selected as the culture material. Somatic embryo induction medium was based on DCR medium supplemented with 0.45 g·L⁻¹ glutamine. -1 CH 0.5g·L -1 Inositol 0.1 g·L -1 PEG 190g·L -1 ABA 8mg·L -1 GA4 mg·L -1 30g / L maltose -1 Aspartic acid 0.2 g·L -1 Proline 0.2 g·L -1 Activated carbon 2g·L -1 Plant-based gel 3g·L -1 The pH was adjusted to 6.0. The final concentration of 5-AzaC was 5 μmol·L⁻¹. -1 10 μmol·L -1 15 μmol·L -1 30 μmol·L -1 and 50 μmol·L -1 Callus tissue was initially cultured on somatic embryonic induction medium without 5-AzaC. All cultures were incubated in the dark at 23°C. After 1, 2, and 3 weeks of initial somatic embryonic induction culture, the materials were transferred to medium containing 5 μmol·L⁻¹. -1 10 μmol·L -1 15 μmol·L -1 30 μmol·L -1 and 50 μmol·L -1 The embryos were cultured in 5-AzaC-containing somatic embryo induction medium to investigate the optimal treatment time and concentration of 5-AzaC.

[0062] After 1, 2, and 3 weeks of initial culture for somatic embryo induction, the material was transferred to 5 μmol·L⁻¹. -1 10 μmol·L -1 15 μmol·L -1 30 μmol·L -1 and 50 μmol·L -1The results were obtained on 5-AzaC somatic embryo induction medium. Figure 4 As shown, considering both whole-plate and single-piece callus tissue, the larger the callus, the lower the degree of material differentiation, i.e., 30–50 μmol·L⁻¹. -1 5-AzaC showed poor induction of somatic embryos. However, somatic embryos treated with 5-AzaC after two weeks of initial culture for induction exhibited better maturation than those after one or three weeks of initial culture, and the embryos were milky white, uniformly distributed, and dense. The experiment indicated that treatment with 5-AzaC after two weeks of initial culture for induction yielded the best results, with the optimal concentration being 5–15 μmol·L⁻¹. -1 It is quite suitable.

[0063] Example 5

[0064] Somatic embryo germination into seedlings: After two weeks of initial culture following the somatic embryo induction method in Example 4, the embryos were transferred to a culture medium containing 10 μmol·L⁻¹. - 1 Mature embryos obtained by culturing in 5-AzaC embryonic induction medium for 7 weeks were used as culture material.

[0065] (1) The somatic embryo germination and seedling culture medium was based on WPM (Cooler; PM1331) supplemented with 30 g·L⁻¹ sucrose. -1 8 g / L agar -1 The pH was adjusted to 5.8. The final concentration of 5-AzaC was set to 0 μmol·L⁻¹. -1 5 μmol·L -1 10 μmol·L -1 and 15 μmol·L -1 The culture medium for somatic embryo germination and seedling formation was added separately. Incandescent lamps were used for illumination, with a duration of 16 hours / day and a light intensity of 260 ± 30 μmol / m². -2 .s -1 At a temperature of 23℃, the embryos were cultured for 30 days, and the embryo formation was observed.

[0066] Adding a low concentration of 5-AzaC to the germination and seedling culture medium resulted in a high seedling rate, reaching up to 95%. The results showed that ( Figure 5 As shown in Table 2), treatment with 5-AzaC in the culture medium resulted in faster somatic embryo seedling development and a higher seedling survival rate than the control. In the control group, callus tissue easily re-formed at the radicle of the somatic embryos, leading to a very low seedling formation rate. Other coniferous species, such as Pinaceae, may use drying treatment to improve the seedling formation rate, but the procedure is cumbersome and the effect is not as good as that of 5-AzaC in this invention. Figure 5 It can be seen that ( Figure 5 From left to right, the final concentrations of 5-AzaC are 0 μmol·L⁻¹. -1 5 μmol·L -110 μmol·L -1 and 15 μmol·L -1 Add 5 μmol·L to the culture medium. -1 ~15μmol·L -1 In 5-AzaC, almost no callus tissue forms around the embryo, and more needles gradually grow. It will eventually root and grow into a seedling.

[0067] Table 2. Statistical results of seedling survival rate

[0068] <![CDATA[0μmol·L -1 ]]> 20 3 15％ <![CDATA[5μmol·L -1 ]]> 20 17 85％ <![CDATA[10μmol·L -1 ]]> 20 19 95％ <![CDATA[15μmol·L -1 ]]> 20 14 70％

[0069] Example 6

[0070] Add 10 μmol·L to the somatic embryo induction medium -1 The 5-AzaC low-embryotype can achieve somatic embryo induction rates from low to high.

[0071] Experimental procedure:

[0072] In the somatic embryo induction experiment, T3 callus tissue subcultured for 10 days (Chen T, Wang D, Yuan G, et al. High-and low-embryogenic Taxodium hybrid 'zhongshanshan' cell lines reveal involvement of redox homeostasis in somatic embryogenesis[J]. Scientia Horticulturae, 2025, 339. DOI:10.1016 / j.scienta.2024.113830.) was inoculated into cells containing 0 and 10 μmol·L⁻¹, respectively. -1 5-AzaC somatic embryo induction medium. The medium was based on DCR basal medium, supplemented with hydrolyzed casein (0.5 g·L⁻¹). -1 Inositol (0.1 g·L) -1 ), polyethylene glycol (190 g·L) -1 (Shanghai Yuanye Biotechnology, catalog number V32174), glutamine (0.45 g·L) -1 ), aspartic acid (0.2 g·L) -1 ), proline (0.2 g·L) -1 ), maltose (30g·L) -1 ), activated carbon (2 g·L) -1 ), plant gel (3g·L) -1 Abscisic acid (ABA, 8 mg·L) -1 ) and gibberellin (GA, 4 mg·L) -1(Shanghai Yuanye Biotechnology, catalog number S28506). After adjusting the pH of the culture medium to 6.0, it was autoclaved at 121℃ for 20 min. ABA, GA, and 5-AzaC, which had undergone aseptic filtration, were added after the culture medium had cooled to 50℃. All cultures were incubated in the dark at 23℃, and the somatic embryo induction effect was observed using a stereomicroscope.

[0073] The results are as follows Figure 6 As shown, at 5 days of somatic embryonic induction, early protoembryos appeared on the surface of callus treated with 5-AzaC, while the proliferation of control callus was very obvious; at 15 days of somatic embryonic induction, the 5-AzaC-treated group had formed dominant embryos, while early protoembryos appeared on the surface of the control group callus; at 40 days of somatic embryonic induction, the 5-AzaC-treated group had more mature cotyledonary embryos, while the control group had only one. Overall, 5-AzaC treatment can promote later differentiation by inhibiting rapid early proliferation of callus, thus enabling low-embryogenic callus to differentiate into more cotyledonary embryos.

[0074] Therefore, this invention, by adding 5-AzaC to the somatic embryo induction medium and germination seedling medium, improves the somatic embryo induction efficiency and accelerates the induction speed of Metasequoia glyptostroboides. It achieves a significant increase in somatic embryo induction rate for low-embryo genotypes and greatly improves the seedling formation rate. Furthermore, the procedure is simple, requiring only the addition of a low concentration of 5-AzaC to the somatic embryo induction or germination medium to increase the somatic embryo induction rate, accelerate the somatic embryogenesis process, and achieve a seedling formation rate of over 95%. In addition, no drying treatment is required, greatly shortening the seedling formation time. This invention is of great significance for the breeding and widespread application of superior Metasequoia glyptostroboides varieties.

[0075] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of DNA methylation inhibitors in promoting somatic embryogenesis of Taxus chinensis, characterized in that, The DNA methylation inhibitor is 5-AzaC, and the concentration of 5-AzaC is 5~15 μmol·L. -1 ; The 5-AzaC reduces the overall methylation level of the genome.

2. A method for promoting somatic embryogenesis in Taxodium distichum, characterized in that, The steps are as follows: The callus tissue of Metasequoia glyptostroboides was initially cultured in a somatic embryo induction medium to obtain cultured Metasequoia glyptostroboides callus tissue. The cultured Metasequoia callus was subjected to somatic embryo induction culture in a somatic embryo induction medium containing DNA methylation inhibitors to obtain mature Metasequoia somatic embryos. The mature somatic embryos of Metasequoia glyptostroboides were cultured in a somatic embryo germination and seedling culture medium containing DNA methylation inhibitors to obtain Metasequoia glyptostroboides somatic embryo seedlings. The DNA methylation inhibitor is 5-AzaC; 5-AzaC reduces the overall methylation level of the genome; The somatic embryo induction medium uses DCR as the basal medium and also adds the following components at concentrations: glutamine 0.3~0.5 g·L⁻¹. -1 CH 0.2~0.5g·L -1 Inositol 0.1~1 g·L -1 PEG 170~210g·L -1 ABA 7~9mg·L -1 GA 3~5mg·L -1 Maltose 25~35g·L -1 Aspartic acid 0.1~0.3 g·L -1 Proline 0.1~0.3 g·L -1 Activated carbon 2~3g·L -1 3~4g·L of plant gel -1 ; The somatic embryo induction medium containing DNA methylation inhibitors uses DCR as the basal medium and also adds the following components at concentrations: glutamine 0.3~0.5 g·L⁻¹. -1 CH 0.2~0.5g·L -1 Inositol 0.1~1 g·L -1 PEG 170~210g·L -1 ABA 7~9mg·L -1 GA 3~5mg·L -1 Maltose 25~35g·L -1 Aspartic acid 0.1~0.3 g·L -1 Proline 0.1~0.3 g·L -1 Activated carbon 2~3g·L -1 Plant-based gel 3~4g·L -1 and 5-AzaC 5~15μmol·L -1 ; The somatic embryo germination and seedling culture medium containing DNA methylation inhibitors uses WPM as the basal medium and also adds the following components at concentrations: sucrose 20-30 g / L. -1 7-8 g / L of agar -1 and 5-AzaC 5~15μmol·L -1 .

3. The method according to claim 2, characterized in that, The initial culture of the somatic embryo induction is a dark culture, the temperature of the initial culture of the somatic embryo induction is 21~25℃, and the time of the initial culture of the somatic embryo induction is 2 weeks.

4. The method according to claim 2, characterized in that, The somatic embryo induction culture is a dark culture, the temperature of the somatic embryo induction culture is 21~25℃, and the time of the somatic embryo induction culture is 6~7 weeks.

5. The method according to claim 2, characterized in that, The photoperiod duration for somatic embryo germination and seedling culture was 16 h / d, and the light intensity was 230–290 μmol / m. -2 .s -1 The temperature for embryo germination and seedling culture is 21~25℃.

6. The method according to claim 2, characterized in that, The Metasequoia callus includes T170 genotype callus and T3 genotype callus.

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