Application of DNA methylation inhibitor in promoting taxodium zhongshanha somatic embryogenesis

Abstract

The invention belongs to the technical field of plant embryogenesis, and particularly relates to application of a DNA methylation inhibitor to promotion of taxodium zhongshanha somatic embryogenesis, and the DNA methylation inhibitor comprises 5-AzaC. According to the method, 5-AzaC is added into the somatic embryo induction culture medium and the somatic embryo germination seedling culture medium, so that the somatic embryo induction efficiency is improved, the induction speed is accelerated, the somatic embryo induction rate is increased from low to high for somatic embryo induction with low embryo genotype, and the somatic embryo seedling rate is greatly increased. Meanwhile, according to the scheme, the operation steps are simple, the somatic embryo induction rate can be increased, the somatic embryo generation process can be accelerated only by adding the 5-AzaC with the low concentration into the somatic embryo induction culture medium or the somatic embryo germination seedling culture medium, and the seedling rate can reach 95% or above. In addition, the method does not need drying treatment, and the seedling forming time is greatly shortened. The method has important significance on breeding, popularization and application of excellent varieties of taxodium Zhongshanha.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant somatic embryogenesis, and particularly relates to the application of DNA methylation inhibitors in promoting the somatic embryogenesis of Taxodium 'zhongshanshan'. Background Art

[0002] Taxodium 'zhongshanshan' is a series of excellent varieties selected from the interspecific hybridization breeding of Taxodium. It has the advantages of fast growth, salt tolerance and waterlogging tolerance, and is widely used in the construction of ecological barriers such as the middle and lower reaches of the Yangtze River and its surrounding lake low-lying wetlands, and the coastal saline-alkali land in the southeast coast of China, producing good ecological, economic and social benefits, and having a huge social demand. All along, the main method for breeding seedlings of excellent clones of Taxodium 'zhongshanshan' is through softwood cutting. However, in the process of cutting propagation, it is found that with the increase of the physiological age of Taxodium 'zhongshanshan' clones and the multi-generation cutting propagation, the cutting rooting rate gradually decreases, and there are significant differences in the rooting ability and rooting quality among different varieties. These problems seriously limit the breeding, popularization and application of excellent varieties of Taxodium 'zhongshanshan'.

[0003] Somatic embryogenesis refers to the process in which somatic cells are induced to form non-zygotic embryos under certain conditions and germinate into complete plants. This technology has the advantages of a large number of propagations and high speed. However, the current somatic embryogenesis system of Taxodium 'zhongshanshan' still has problems such as low somatic embryogenesis efficiency and large differences in somatic embryogenesis frequencies among different strains, and it is necessary to further optimize the somatic embryogenesis system of Taxodium 'zhongshanshan'. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of DNA methylation inhibitors in promoting the somatic embryogenesis of Taxodium 'zhongshanshan'. By adding a lower concentration of 5-AzaC to the somatic embryo induction or somatic embryo germination and seedling formation medium in the present invention, the somatic embryo induction rate can be increased, the somatic embryogenesis process can be accelerated, and the seedling formation rate can reach more than 95%.

[0005] The present invention provides the application of DNA methylation inhibitors in promoting the somatic embryogenesis of Taxodium 'zhongshanshan', and the DNA methylation inhibitors include 5-AzaC.

[0006] The present invention also provides a method for promoting the somatic embryogenesis of Taxodium 'zhongshanshan', including the following steps:

[0007] Carrying out primary somatic embryo induction culture on Taxodium 'zhongshanshan' callus in a somatic embryo induction medium to obtain the cultured Taxodium 'zhongshanshan' callus;

[0008] Carrying out somatic embryo induction culture on the cultured Taxodium 'zhongshanshan' callus in a somatic embryo induction medium containing a DNA methylation inhibitor to obtain mature somatic embryos of Taxodium 'zhongshanshan';

[0009] Carrying out somatic embryo germination and seedling formation culture on the mature somatic embryos of Taxodium 'zhongshanshan' in a somatic embryo germination and seedling formation medium containing a DNA methylation inhibitor to obtain Taxodium 'zhongshanshan' somatic embryo seedlings.

[0010] As a preferred embodiment, the somatic embryo induction medium is based on DCR and further comprises components with the following concentrations: glutamine 0.3 - 0.5 g·L -1 , CH 0.2 - 0.5 g·L -1 , inositol 0.1 - 1 g·L -1 , PEG 170 - 210 g·L -1 , ABA 7 - 9 mg·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 somatic embryo induction culture is a dark culture, the temperature of the initial somatic embryo induction culture is 21 - 25 °C, and the time of the initial somatic embryo induction culture is 2 weeks.

[0012] As a preferred embodiment, the DNA methylation inhibitor is 5 - AzaC, and the concentration of 5 - AzaC during somatic embryo induction culture is 5 - 15 μmol·L -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 °C, and the time of the somatic embryo induction culture is 6 - 7 weeks.

[0014] As a preferred embodiment, the somatic embryo germination and seedling - forming medium is based on WPM and further comprises components with the following concentrations: sucrose 20 - 30 g·L -1 and agar 7 - 8 g·L -1 .

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

[0016] As a preferred embodiment, the light duration of the somatic embryo germination and seedling - forming culture is 16 h / d, the light intensity of the somatic embryo germination and seedling - forming culture is 230 - 290 μmol·m -2 .s -1 , and the temperature of the somatic embryo germination and seedling - forming culture is 21 - 25 °C.

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

[0018] Beneficial effects: The present invention provides the application of a DNA methylation inhibitor in promoting somatic embryogenesis of Taxodium hybrid, and 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, the somatic embryo induction efficiency of Taxodium hybrid is improved, the induction speed is accelerated, the somatic embryo induction rate of low-embryo genotypes is increased from low to high, and the somatic embryo seedling formation rate is greatly improved. At the same time, the operation steps of the present invention are simple. Only by adding a low concentration of 5-AzaC to the somatic embryo induction medium or the somatic embryo germination and seedling formation medium, the somatic embryo induction rate can be increased, the somatic embryogenesis process can be accelerated, and the seedling formation rate can reach more than 95%. In addition, the method of the present invention does not require drying treatment, and the seedling formation time is greatly shortened. It is of great significance for the breeding, popularization and application of excellent varieties of Taxodium hybrid. Description of the drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0020] Figure 1 It is a microscopic structure comparison diagram of the callus in Example 1, where the concentrations of 5-AzaC in a-f and (a)-(f) are 0 μmol·L 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 , where a-f are the comparison diagrams of the whole dish, and (a)-(f) are the microscopic structure comparisons of the callus;

[0021] Figure 2 It is the influence of different concentrations of 5-AzaC on the proliferation of callus in Example 1;

[0022] Figure 3 It is the influence of different concentrations of 5-AzaC on the number of mature somatic embryos in Example 3;

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

[0024] Figure 5 For the effect of different concentrations of 5-AzaC in Example 5 on the germination and seedling formation of mature somatic embryos, the concentrations of 5-AzaC from left to right are 0 μmol·L -1 , 5 μmol·L -1 , 10 μmol·L -1 and 15 μmol·L -1 ;

[0025] Figure 6 For the effect of 5-AzaC in Example 6 on somatic embryo induction of T3 genotype callus. Specific implementation method

[0026] The present invention provides the application of DNA methylation inhibitors in promoting Taxodium hybridum somatic embryogenesis, and the DNA methylation inhibitors include 5-AzaC.

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

[0028] Culturing Taxodium hybridum callus in a somatic embryo induction medium for primary somatic embryo induction to obtain cultured Taxodium hybridum callus;

[0029] Culturing the cultured Taxodium hybridum callus in a somatic embryo induction medium containing a DNA methylation inhibitor for somatic embryo induction culture to obtain mature Taxodium hybridum somatic embryos;

[0030] Culturing the mature Taxodium hybridum somatic embryos in a somatic embryo germination and seedling formation medium containing a DNA methylation inhibitor for somatic embryo germination and seedling formation culture to obtain Taxodium hybridum somatic embryo seedlings.

[0031] As a specific implementation method, the Taxodium hybridum callus can be selected as the Taxodium hybridum callus subcultured for 10 days, and the subculture medium can be selected as a callus proliferation blank medium: using DCR as the basic medium, supplemented with glutamine 0.45 g·L -1 , CH (hydrolyzed casein) 0.5 g·L -1 , inositol 0.1 g·L -1 , NAA (naphthaleneacetic acid) 2 mg·L -1 , maltose 20 g·L -1 , activated carbon 2.5 g·L -1 , plant gel 2.8 g·L -1 , and the pH is adjusted to 5.8.

[0032] The somatic embryo induction medium of the present invention uses DCR as the basic medium and further includes the following components at the following concentrations: glutamine 0.3 - 0.5 g·L-1 、 Casein Hydrolysate (CH) 0.2 - 0.5 g·L -1 、 Inositol 0.1 - 1 g·L -1 、 Polyethylene Glycol (PEG) 170 - 210 g·L -1 、 Abscisic Acid (ABA) 7 - 9 mg·L -1 、 Gibberellic Acid (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 Phytagel 3 - 4 g·L -1 。 As a specific embodiment, the somatic embryo induction medium is based on DCR medium and further includes the following components at the following concentrations: Glutamine 0.45 g·L -1 、 CH 0.5 g·L -1 、 Inositol 0.1 g·L -1 、 PEG 190 g·L -1 、 ABA 8 mg·L -1 、 GA 4 mg·L -1 、 Maltose 30 g·L -1 、 Aspartic Acid 0.2 g·L -1 、 Proline 0.2 g·L -1 、 Activated Carbon 2 g·L -1 and Phytagel 3 g·L -1 , The pH is adjusted to 6.0; As another specific embodiment, the somatic embryo induction medium is based on DCR medium and further includes the following components at the following concentrations: Glutamine 0.3 g·L -1 、 CH 0.2 g·L -1 、 Inositol 0.1 g·L -1 、 PEG 170 g·L -1 、 ABA 7 mg·L -1 、 GA 3 mg·L -1 、 Maltose 25 g·L -1 、 Aspartic Acid 0.1 g·L -1 、 Proline 0.1 g·L -1 、 Activated Carbon 2 g·L -1 and Phytagel 3 g·L -1 , The pH is adjusted to 6.0; As another specific embodiment, the somatic embryo induction medium is based on DCR medium and further includes the following components at the following concentrations: Glutamine 0.5 g·L -1 、 CH 0.5 g·L -1 、 Inositol 1 g·L -1, PEG 210 g·L -1 , ABA 9 mg·L -1 , GA 5 mg·L -1 , maltose 35 g·L -1 , aspartic acid 0.3 g·L -1 , proline 0.3 g·L -1 , activated carbon 3 g·L -1 and phytagel 4 g·L -1 , adjust the pH to 6.0.

[0033] The primary somatic embryo induction culture described in the present invention is a dark culture. The temperature of the primary somatic embryo induction culture is 21 - 25 °C, and the time of the primary somatic embryo induction culture is 2 weeks. As a specific embodiment, the temperature of the primary somatic embryo induction culture can be 21 °C, 22 °C, 23 °C, 24 °C or 25 °C. The examples of the present invention show that when the time of the primary somatic embryo induction culture is 2 weeks, the somatic embryo maturation effect after 5-AzaC treatment is better than that when the time of the primary somatic embryo induction culture is 1 week and 3 weeks. When the time of the primary somatic embryo induction culture is 2 weeks, the somatic embryos are milky white, evenly and densely distributed.

[0034] The DNA methylation inhibitor described in the present invention is 5-AzaC, and the concentration of 5-AzaC during somatic embryo induction culture is 5 - 15 μmol·L -1 . As a specific embodiment, the concentration of 5-AzaC can be 5 μmol·L -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 the present invention, 5-AzaC (5-azacytidine) as a DNA methylation inhibitor can inhibit the activity of DNA methyltransferase and reduce the overall genomic methylation level. The role of 5-AzaC in the present invention may be to relieve the silencing state of certain genes (such as key genes for embryo 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 of the present invention is dark culture, the temperature of the somatic embryo induction culture is 21-25 °C, and the time of the somatic embryo induction culture is 6-7 weeks. As a specific implementation manner, the temperature of the somatic embryo induction culture can be 21 °C, 22 °C, 23 °C, 24 °C or 25 °C. As a specific implementation manner, the time of the somatic embryo induction culture can be 6 weeks or 7 weeks.

[0036] The somatic embryo germination and seedling formation medium of the present invention uses WPM as the basic medium and further includes components with the following concentrations: sucrose 20-30 g·L -1 and agar 7-8 g·L -1 . As a specific implementation manner, the somatic embryo germination and seedling formation medium uses WPM as the basic medium and further includes components with the following concentrations: sucrose 30 g·L -1 and agar 8 g·L -1 , and the pH is adjusted to 5.8; as another specific implementation manner, the somatic embryo germination and seedling formation medium uses WPM as the basic medium and further includes components with the following concentrations: sucrose 20 g·L -1 and agar 7 g·L -1 , and the pH is adjusted to 5.8; as another specific implementation manner, the somatic embryo germination and seedling formation medium uses WPM as the basic medium and further includes components with the following concentrations: sucrose 25 g·L -1 and agar 7.3 g·L -1 , and the pH is adjusted to 5.8; as another specific implementation manner, the somatic embryo germination and seedling formation medium uses WPM as the basic medium and further includes components with the following concentrations: sucrose 28 g·L -1 and agar 7.5 g·L -1 , and the pH is adjusted to 5.8.

[0037] The concentration of 5-AzaC during the somatic embryo germination and seedling formation culture of the present invention is 5-15 μmol·L -1 . As a specific implementation manner, the concentration of 5-AzaC can be 5 μmol·L -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 manner, when the concentration of 5-AzaC in the somatic embryo germination and seedling formation culture is 10 μmol·L-1 The seedling formation rate is the highest at this time, reaching up to 95%.

[0038] The light duration for the somatic embryo germination and seedling formation culture in the present invention is 16 h / d, and the light intensity for the somatic embryo germination and seedling formation culture is 230 - 290 μmol·m -2 .s -1 , and the temperature for the somatic embryo germination and seedling formation culture is 21 - 25 °C. As a specific implementation manner, the light intensity can be 230 μmol·m -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 . As a specific implementation manner, the temperature for the somatic embryo germination and seedling formation culture can be 21 °C, 22 °C, 23 °C, 24 °C or 25 °C.

[0039] The Taxodium hybrid callus in the present invention includes T170 genotype callus and T3 genotype callus. As a specific implementation manner, the T3 genotype callus is a low-embryo genotype callus, that is, the embryo formation rate of this genotype is extremely low. In the present invention, by adding 10 μmol·L -1 of 5-AzaC to the somatic embryo induction medium, the transformation of the somatic embryo induction rate of the low-embryo genotype T3 genotype callus from low to high is realized, and more cotyledon embryos are differentiated from the low-embryonic callus.

[0040] In the present invention, 5-AzaC is added to the somatic embryo induction medium and the somatic embryo germination and seedling formation medium, which improves the somatic embryo induction efficiency of Taxodium hybrid, speeds up the induction speed, realizes the transformation of the somatic embryo induction rate of the low-embryo genotype from low to high, and greatly improves the somatic embryo seedling formation rate. At the same time, the operation steps of the scheme of the present invention are simple. Only by adding a low concentration of 5-AzaC to the somatic embryo induction medium or the somatic embryo germination and seedling formation medium, the somatic embryo induction rate can be improved, the somatic embryo development process can be accelerated, and the seedling formation rate can reach more than 95%. In addition, the method of the present invention does not require drying treatment, and the seedling formation time is greatly shortened. It has important significance for the breeding and popularization of excellent varieties of Taxodium hybrid.

[0041] To further illustrate the present invention, the following examples are used to describe in detail the application of the DNA methylation inhibitor provided by the present invention in promoting somatic embryogenesis of Taxodium hybrid 'zhongshanshan', but they should not be construed as limiting the protection scope of the present invention.

[0042] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products well-known to those skilled in the art can be used.

[0043] Example 1

[0044] Callus of T170 genotype (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: For the callus proliferation blank medium (using DCR (Coolaber; product number: PM1631) as the basic medium, supplemented with glutamine 0.45 g·L -1 , CH 0.5 g·L -1 , inositol 0.1 g·L -1 , NAA 2 mg·L -1 , maltose 20 g·L -1 , activated carbon 2.5 g·L -1 , gel 2.8 g·L -1 ), after adjusting the pH value of the medium to 5.8, it was sterilized at 121 °C for 20 min. After the medium cooled to 60 °C, sterile 5-AzaC (final concentrations were 0 μmol·L -1 , 5 μmol·L -1 , 10 μmol·L -1 , 15 μmol·L -1 , 30 μmol·L -1 and 50 μmol·L -1 ) that had been filtered was added to it. After 10 days of subculture of the embryogenic callus of T170 genotype, callus that was growing vigorously and in a transparent state was selected and inoculated into media containing 0 μmol·L -1 , 5 μmol·L -1 , 10 μmol·L -1, 15 μmol·L -1 , 30 μmol·L -1 and 50 μmol·L -1 in the subculture medium of 5-azacytidine (5-AzaC, Shanghai Aladdin, catalog number A100625), that is, the callus proliferation blank medium containing different concentrations of 5-AzaC. All cultures were placed in an incubator under dark conditions and incubated at a constant temperature of 23°C. After 15 days of culture, the fresh weight of embryogenic callus was weighed. Subsequently, a small amount of callus sample was placed on a glass slide, distilled water was added, and it was gently dispersed with forceps. A coverslip was covered, and the cell morphology was observed under a microscope.

[0046] In this experiment, at least 3 replicate culture dishes were set for each treatment, and 6 pieces of callus about 0.2 g were inoculated in each dish. The initial fresh weight of the callus at the time of inoculation was recorded as W0, and the fresh weight of the callus after culture was W1. The formula for calculating the fresh weight increase of callus proliferation is W1 - W0, and the proliferation rate is calculated according to the formula (W1 - W0) / W0 × 100%.

[0047] Result analysis:

[0048] The effects of different concentrations of 5-AzaC on the proliferation of T170 genotype callus are shown in Figure 1 , (from left to right, the concentrations are 0 μmol·L -1 , 5 μmol·L -1 , 10 μmol·L -1 , 15 μmol·L -1 , 30 μmol·L -1 and 50 μmol·L -1 ). The upper part is the comparison picture of the whole dish, and the lower part is the comparison of the microscopic structure of the callus. The results show that in the study of embryogenic callus proliferation, compared with the control group, the 5-AzaC treatment significantly inhibited its proliferation effect. The volume of the embryogenic callus in the control group ( Figure 1 a) was significantly larger than that of each 5-AzaC treatment group ( Figure 1 b-f). Morphological evaluation showed that the control group ( Figure 1 a) and the 5-AzaC treatment group ( Figure 1 b-f) both showed a similar white and transparent appearance, with a moist surface and no significant structural differences ( Figure 1 (a)-(f)).

[0049] Through statistical analysis, it was found that ( Figure 2 ), as the concentration of 5-AzaC increased from the control group (0 μmol·L -1 ) to 50 μmol·L -1The proliferation rate of embryogenic callus decreased significantly from the initial about 350% to about 30% (P<0.05). It can be seen from this that the addition of 5-AzaC has a significant inhibitory effect on the proliferation of embryogenic callus.

[0050] Example 2

[0051] Select the callus of T170 genotype subcultured for 10 d as the culture material. The somatic embryo induction medium uses DCR (Coolaber; product number: PM1631) as the basic medium, supplemented with 0.45 g·L -1 of glutamine, 0.5 g·L -1 of CH (casein hydrolysate), 0.1 g·L -1 of inositol, 190 g·L -1 of PEG (polyethylene glycol) (Shanghai Yuanye Bio-Technology Co., Ltd., product number V32174), 8 mg·L -1 of ABA (abscisic acid), 4 mg·L -1 of GA (gibberellin) (Shanghai Yuanye Bio-Technology Co., Ltd., product number S28506), 30 g·L -1 of aspartic acid, 0.2 g·L -1 of proline, 0.2 g·L -1 of activated carbon, 2 g·L -1 of plant gel, 3 g·L -1 , and the pH is adjusted to 6.0. Set the final concentration of 5-AzaC to 0 μmol·L -1 , 5 μmol·L -1 , 10 μmol·L -1 and 15 μmol·L -1 , and add them to the somatic embryo induction medium respectively. Record the somatic embryo occurrence after culturing for 60 d, and calculate the number of mature embryos obtained by induction. The somatic embryo induction is dark culture, and the temperature is 23 °C.

[0052] The results are shown in Table 1. It was found that the treatment with low concentration of 5-AzaC accelerated the process of somatic embryo occurrence, while the treatment with high concentration of 5-AzaC inhibited the efficiency of somatic embryo occurrence. Mature cotyledon embryos appeared in the CK treatment at the 6th week. Mature cotyledon embryos appeared in the 5-AzaC 5 μmol·L -1 and 10 μmol·L -1 treatments at the 5th week, 1 week earlier than CK.

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

[0054]

[0055] Example 3

[0056] In the somatic embryo induction experiment, the callus of T170 genotype subcultured for 10 days was selected as the culture material, and the callus subcultured for 10 days was inoculated on the somatic embryo induction medium containing 0 μmol·L -1 , 5 μmol·L -1 , 10 μmol·L -1 , 15 μmol·L -1 , 30 μmol·L -1 and 50 μmol·L -1 of 5-AzaC. The medium was based on DCR basal medium, supplemented with casein hydrolysate (0.5 g·L -1 ), inositol (0.1 g·L -1 ), polyethylene glycol (190 g·L -1 ) (Shanghai Yuanye Bio-Technology Co., Ltd., product number V32174), glutamine (0.45 g·L -1 ), aspartic acid (0.2 g·L -1 ), proline (0.2 g·L -1 ), maltose (30 g·L -1 ), activated carbon (2 g·L -1 ), phytagel (3 g·L -1 ), abscisic acid (ABA, 8 mg·L -1 ) and gibberellin (GA, 4 mg·L -1 ) (Shanghai Yuanye Bio-Technology Co., Ltd., product number S28506). After adjusting the pH value of the medium to 6.0, the remaining components were autoclaved at 121 °C for 20 min. Among them, the aseptically filtered ABA, GA and 5-AzaC needed to be added after the medium cooled to 60 °C. All cultures were placed in an incubator and incubated at a constant temperature of 23 °C in the dark. The tissue structure was observed under a microscope every 5 days. At least 3 replicate culture dishes were set for each treatment, and 6 pieces of embryogenic callus about 0.2 g were inoculated in each dish. After 60 days of culture, the number of columnar embryos (N1) and mature embryos (N2) was counted, and the somatic embryo maturation rate was calculated according to the formula somatic embryo maturation rate = N2 / (N1 + N2) × 100%.

[0057] Result analysis:

[0058] From the perspective of somatic embryo induction efficiency ( Figure 3 ), the number of mature cotyledon embryos treated with 5 μmol·L -1 , 10 μmol·L -1 and 15 μmol·L -1 of 5-AzaC increased, and the maturation rate of the treatment with 10 μmol·L -1 was the highest, with about 9 mature somatic embryos per dish. The treatments with 30 μmol·L -1 and 50 μmol·L -1Treatment with 5-AzaC led to slower SE development, a lower maturation rate compared to the control, and the number of mature somatic embryos per dish was approximately 5 and 3, respectively.

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

[0060] Example 4

[0061] Callus of T170 genotype subcultured for 10 d was selected as the culture material. The somatic embryo induction medium was based on DCR medium, supplemented with 0.45 g·L -1 of glutamine, 0.5 g·L -1 of CH, 0.1 g·L -1 of inositol, 190 g·L -1 of PEG, 8 mg·L -1 of ABA, 4 mg·L -1 of GA, 30 g·L -1 of maltose, 0.2 g·L -1 of aspartic acid, 0.2 g·L -1 of proline, 2 g·L -1 of activated carbon, 3 g·L -1 of plant gel, and the pH was adjusted to 6.0. The final concentrations of 5-AzaC were 5 μmol·L -1 、10 μmol·L -1 、15 μmol·L -1 、30 μmol·L -1 and 50 μmol·L -1 . The callus was first subjected to primary somatic embryo induction culture on the somatic embryo induction medium without 5-AzaC. All cultures were placed in an incubator under dark conditions and cultured at a constant temperature of 23 °C. After 1 week, 2 weeks, and 3 weeks of primary somatic embryo induction culture, the materials were transferred to the somatic embryo induction medium containing 5 μmol·L -1 、10 μmol·L -1 、15 μmol·L -1 、30 μmol·L -1 and 50 μmol·L -1 of 5-AzaC for continued culture to study the optimal treatment time and concentration of 5-AzaC.

[0062] After 1 week, 2 weeks, and 3 weeks of primary somatic embryo induction culture, respectively, the materials were transferred to 5 μmol·L -1 、10 μmol·L -1 、15 μmol·L -1 、30 μmol·L -1 and 50 μmol·L -1on the somatic embryo induction medium of 5-AzaC. The results are as Figure 4 shown. From the perspective of the whole dish and single callus, the larger the callus piece, the lower the degree of material differentiation, that is, 30-50 μmol·L -1 The somatic embryo induction effect of 5-AzaC is not good. And the somatic embryo maturation effect of treating with 5-AzaC 2 weeks after the initial culture of somatic embryo induction is better than that of the somatic embryos 1 week and 3 weeks after the initial culture of somatic embryo induction, and the somatic embryos are milky white, evenly and densely distributed. The experiment shows that the treatment effect of 5-AzaC is the best 2 weeks after the initial culture of somatic embryo induction, and the addition concentration is set at 5-15 μmol·L -1 is more appropriate.

[0063] Example 5

[0064] Germination of somatic embryos into seedlings: Using the mature somatic embryos obtained by culturing for 7 weeks in the somatic embryo induction medium containing 10 μmol·L - 1 5-AzaC after 2 weeks of the initial culture of somatic embryo induction in Example 4 as the culture material.

[0065] (1) The somatic embryo germination and seedling medium uses WPM (Coolaber; PM1331) as the basic medium, supplemented with 30 g·L -1 of sucrose, 8 g·L -1 of agar, and the pH is adjusted to 5.8. The final concentration of 5-AzaC is set to 0 μmol·L -1 , 5 μmol·L -1 , 10 μmol·L -1 and 15 μmol·L -1 are respectively added to the somatic embryo germination and seedling medium. The light source is incandescent light, the light time is 16 h / d, the light intensity is 260±30 μmol·m -2 .s -1 , the temperature is 23 °C, and the somatic embryo seedling formation is observed after culturing for 30 d.

[0066] Adding a low concentration of 5-AzaC to the germination and seedling medium has a relatively high seedling formation rate, up to 95%. The research results show ([[]] Figure 5 and Table 2), adding 5-AzaC treatment to the medium, the somatic embryo seedling development is faster, and the seedling survival rate is higher than that of the control. In the control group, callus is extremely likely to be generated again at the radicle of the somatic embryo, which will lead to a very low somatic embryo seedling formation rate. For other coniferous tree species, such as Pinaceae, the drying treatment method is used, which can improve the somatic embryo seedling formation rate, but the operation steps are cumbersome and the effect is not as good as that of 5-AzaC in the present invention. From Figure 5 it can be seen ([[]] Figure 5 from left to right in [], the final concentrations of 5-AzaC are 0 μmol·L -1 , 5 μmol·L -1, 10 μmol·L -1 and 15 μmol·L -1 ), 5 μmol·L -1 to 15 μmol·L -1 of 5-AzaC was added to the medium, and almost no callus formed around the somatic embryos, and more needles gradually grew out, and then they would be cultured to gradually take root and form seedlings.

[0067] Table 2 Statistical results of seedling formation rate

[0068] 5-AzaC concentration Number of inoculated mature somatic embryos Number of seedlings Seedling rate <![CDATA[0 μmol·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] 10 μmol·L -1 of 5-AzaC was added to the somatic embryo induction medium, and the low embryo genotype could achieve the somatic embryo induction rate from low to high

[0071] Experimental procedure:

[0072] In the somatic embryo induction experiment, the subcultured T3 callus for 10 d (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 the somatic embryo induction medium containing 0 and 10 μmol·L -1 of 5-AzaC. The medium was based on the DCR basal medium and added casein hydrolysate (0.5 g·L -1 ), inositol (0.1 g·L -1 ), polyethylene glycol (190 g·L -1 ) (Shanghai Yuanye Bio-Technology Co., Ltd., product number V32174), glutamine (0.45 g·L -1 ), aspartic acid (0.2 g·L -1 ), proline (0.2 g·L -1 ), maltose (30 g·L -1 ), activated carbon (2 g·L -1 ), plant gel (3 g·L -1 ), abscisic acid (ABA, 8 mg·L -1 ) and gibberellin (GA, 4 mg·L -1)(Shanghai Yuanye Bio-Technology Co., Ltd., product number S28506). After adjusting the pH value of the culture medium to 6.0, it was autoclaved at 121 °C for 20 min. Among them, aseptically filtered ABA, GA and 5-AzaC needed to be added after the culture medium was cooled to 50 °C. All cultures were placed in an incubator and incubated at a constant temperature of 23 °C under dark conditions, and the somatic embryo induction effect was observed through a stereomicroscope.

[0073] The results are as Figure 6 shown. When somatic embryo induction was carried out for 5 days, early proembryos had appeared on the surface of the callus treated with 5-AzaC, while the proliferation of the control callus was very obvious; when somatic embryo induction was carried out for 15 days, the 5-AzaC treatment group had formed dominant embryos, while early proembryos had appeared on the surface of the control callus; when somatic embryo induction was carried out for 40 days, the 5-AzaC treatment group had more mature cotyledon embryos, while the control group had only one. Generally speaking, the 5-AzaC treatment would inhibit the rapid proliferation in the early stage of callus, thus being beneficial to the later differentiation, enabling the low-embryogenic callus to differentiate into more cotyledon embryos.

[0074] Thus, it can be seen that adding 5-AzaC to the somatic embryo induction medium and the germination and seedling formation medium of the present invention improves the somatic embryo induction efficiency of Taxodium zhongshanense, speeds up the induction speed, realizes the somatic embryo induction rate from low to high for the somatic embryo induction of low-embryo genotypes, and greatly improves the somatic embryo seedling formation rate. At the same time, the operation steps of the scheme of the present invention are simple. Only by adding a low concentration of 5-AzaC to the somatic embryo induction or germination medium, the somatic embryo induction rate can be increased, the somatic embryo development process can be accelerated, and the seedling formation rate can reach more than 95%. In addition, no drying treatment is required, and the seedling formation time is greatly shortened. It is of great significance for the breeding, popularization and application of excellent varieties of Taxodium zhongshanense.

[0075] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. The use of a DNA methylation inhibitor in promoting embryogenesis of Cunninghamia lanceolata, characterized in that: The DNA methylation inhibitors include 5-AzaC.

2. A method for promoting somatic embryogenesis of Zhongshan fir, characterized in that: The following steps are involved: performing initial somatic embryo induction culture on the callus of Cunninghamia lanceolata in a somatic embryo induction medium to obtain cultured Cunninghamia lanceolata callus; performing somatic embryo induction culture on the cultured C. zhongshanensis callus in a somatic embryo induction medium containing a DNA methylation inhibitor to obtain mature somatic embryos of C. zhongshanensis; The mature somatic embryos of Zhongshan fir are cultured in a somatic embryo germination and seedling formation medium containing a DNA methylation inhibitor to obtain Zhongshan fir somatic embryo seedlings.

3. The method according to claim 2, characterized in that The somatic embryo induction medium uses DCR as the basic medium and also includes the following components at the following concentrations: 0.3-0.5 g·L glutamine -1 、CH 0.2~0.5g·L -1 , inositol 0.1~1g·L -1 、PEG 170~210g·L -1 ,ABA 7~9mg·L -1 GA 3~5mg·L -1 , maltose 25~35g·L -1 , Aspartic acid 0.1~0.3g·L -1 , Proline 0.1~0.3g·L -1 , activated carbon 2~3g·L -1 and plant gel 3-4g·L -1 .

4. The method according to claim 2, characterized in that: The initial culture of somatic embryo induction is dark culture, the temperature of the initial culture of somatic embryo induction is 21-25° C., and the time of the initial culture of somatic embryo induction is 2 weeks.

5. The method according to claim 2, characterized in that: The DNA methylation inhibitor is 5-AzaC, and the concentration of 5-AzaC during somatic embryo induction culture is 5 to 15 μmol·L -1 .

6. The method according to claim 2, characterized in that The somatic embryo induction culture is dark culture, the temperature of the somatic embryo induction culture is 21-25° C., and the time of the somatic embryo induction culture is 6-7 weeks.

7. The method according to claim 2, characterized in that The somatic embryo germination seedling medium uses WPM as the basic medium and also includes the following components at the following concentrations: sucrose 20-30 g·L -1 and agar 7~8g·L -1 .

8. The method according to claim 5, characterized in that The concentration of 5-AzaC during somatic embryo germination and seedling culture is 5 to 15 μmol·L -1 .

9. The method according to claim 2, characterized in that: The illumination time of the somatic embryo germination and seedling culture is 16h / d, and the illumination intensity of the somatic embryo germination and seedling culture is 230-290μmol.m -2 .s -1 The temperature for culturing the somatic embryos for germination and seedling formation is 21-25°C.

10. The method according to claim 2, characterized in that The Zhongshan fir callus includes T170 genotype callus and T3 genotype callus.

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