Recovery method of pinus massoniana embryonic cells based on different subculture times

Abstract

The present application relates to the biological field, and is based on the pine embryo cell regeneration method of different subculture times, based on the spatiotemporal dynamics of ROS level induced in plant tissue culture, the upstream and downstream substances generated by ROS are used to regulate ROS, which can affect the tissue culture effect of plants, and it is found in the research of the present application that different subculture times have different effects on the regeneration ability of pine embryo cells, different pine embryo cells are classified according to different subculture times, and then the use of a series of optimized ROS scavengers can effectively improve the recession phenomenon of pine embryo cells, and provides strong support for the construction of high-efficiency pine somatic embryo seedling raising technology system.

Description

[Technical Field]

[0001] This invention relates to the field of biotechnology, and in particular to a method for the regeneration of embryonic cells from Pinus massoniana based on different number of subcultures. [Background Technology]

[0002] Masson pine (Pinus massoniana Lamb.) is a major afforestation species in southern my country, serving both ecological construction and resin production, and plays a crucial role in forestry ecology and economic development. However, the long breeding cycle of Masson pine, coupled with the aging of mother trees in seed orchards in recent years, has led to decreased fruit production and a shortage of superior varieties, resulting in significant genetic differentiation differences, low stand quality, and low productivity. Furthermore, the increasingly severe pine wilt disease has prompted many regions across the country to issue "pine restriction orders," significantly hindering the development of the Masson pine industry. The innovation and industrial application of fast-growing, highly resistant, and high-yielding superior germplasm are key to promoting the rapid development of the Masson pine industry. Tissue culture technology is the prerequisite and guarantee for efficient germplasm innovation and propagation. Plant regeneration through somatic embryogenesis in tissue culture is characterized by large numbers, rapid propagation, intact structure, and high plant regeneration rate. Especially when combined with liquid nitrogen cryopreservation technology, it has unlimited potential for the propagation of specific superior genotypes, making it the most commonly used seedling propagation method internationally. Somatic embryos are also considered ideal materials for transgenic breeding.

[0003] Coniferous species are notoriously difficult to cultivate in vitro, and somatic embryogenesis is even more challenging for pine species compared to other conifers. Since Huang Jianqiu et al. first reported the regeneration of Masson pine plants through somatic embryogenesis in 1995, numerous scientists in my country have conducted research and development on somatic embryogenesis technology for Masson pine, but large-scale production and application of Masson pine somatic embryo seedlings have not yet been achieved. Studies have shown that apoptosis of embryogenic cells in Masson pine is significant during subculture propagation, growth viability declines rapidly, and browning and waterlogging are severe, leading to severe subculture degradation and low somatic embryo germination rates. To effectively promote the innovation and efficient propagation of highly resistant Masson pine germplasm, it is essential to develop an efficient somatic embryogenesis technology for Masson pine.

[0004] Reactive oxygen species (ROS) are a class of oxygen-containing chemical substances produced in plants by normal physiological and biochemical activities. They are chemically active and possess strong oxidizing properties, damaging proteins, membrane lipids, and other cellular components, disrupting plant cell integrity, and closely related to apoptosis, pathological changes, and aging. ROS production in plants is closely related to homeostasis; any fluctuation in the intracellular environment can lead to rapid ROS production. Under normal growth conditions, ROS production and clearance in plants maintain a dynamic balance. However, under biotic or abiotic stress, this balance is disrupted, leading to a surge in ROS production and oxidative damage. Compared to normal plant growth, plant tissue culture represents a complex stress, inducing the production of large amounts of ROS. Through long-term natural selection, plants have developed an effective ROS defense mechanism through enzymatic and non-enzymatic systems.

[0005] Currently, there is no research on the impact of upstream and downstream substances generated by ROS on pine somatic embryo culture. Therefore, it is necessary to conduct a systematic study on upstream and downstream substances generated by ROS to accurately identify the culture medium components suitable for the growth of pine somatic embryos. [Summary of the Invention]

[0006] In view of the above, it is necessary to conduct a systematic study on the upstream and downstream substances generated by ROS and accurately identify the culture medium components suitable for the tissue culture growth of pine trees.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for regenerating pine embryonic cells based on different subculture numbers is as follows: Pine embryonic cells are classified according to different subculture numbers into first-generation, second-generation, and third-generation embryonic cells; the first-generation embryonic cells have been subcultured less than 10 times, the second-generation embryonic cells have been subcultured 10 to 30 times, and the third-generation embryonic cells have been subcultured 30 times; GSH, Ca(NO3)2·4H2O, and / or CHX are added to the basal culture medium according to the different subtypes to obtain the corresponding regeneration medium; and the pine embryonic cells are regenerated using the regeneration medium.

[0009] Furthermore, the regeneration medium for the first subgeneration embryonic cells contained 0.5 mM / L GSH and 278 mg / L Ca(NO3)2·4H2O.

[0010] Furthermore, the regeneration medium for the second generation of embryonic cells contained 1 mM / L GSH and 139 mg / L Ca(NO3)2·4H2O.

[0011] Furthermore, the regeneration medium for the third generation of embryonic cells contains 1 mM / L GSH, 139 mg / L Ca(NO3)2·4H2O and 10 mg / L CHX.

[0012] Furthermore, the culture medium consists of the following components: DCR medium without Ca(NO3)2·4H2O, 30 g / L sucrose, 3.5 g / L plant gel, 2 g / L 2,4-D, 0.5 g / L 6-BA, 0.5 g / L acid-hydrolyzed casein and 0.45 g / L glutamine.

[0013] The plant gel used in this application is G1910-Gelrite produced by Sigma-Aldrich, CAS No. 71010-52-1.

[0014] The Ca(NO3)2·4H2O-free DCR medium consists of the following components: 400 mg / L ammonium nitrate, 340 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 370 mg / L magnesium sulfate heptahydrate, 85 mg / L calcium chloride dihydrate, 6.2 mg / L boric acid, 22.3 mg / L manganese sulfate hydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.25 mg / L copper sulfate pentahydrate, 0 Potassium iodide at 0.83 mg / L, cobalt chloride hexahydrate at 0.025 mg / L, lithium chloride at 0.025 mg / L, sodium molybdate dihydrate at 0.25 mg / L, ferrous sulfate heptahydrate at 27.8 mg / L, disodium EDTA at 37.3 mg / L, thiamine hydrochloride at 1.0 mg / L, pyridoxine hydrochloride at 0.5 mg / L, nicotinic acid at 0.5 mg / L, glycine at 2.0 mg / L, and inositol at 200 mg / L.

[0015] The present invention has the following beneficial effects:

[0016] The production of reactive oxygen species (ROS) in plants is closely related to the homeostasis of the internal environment. Any fluctuations in the intracellular environment can lead to a rapid increase in ROS production. Under normal growth conditions, the production and clearance of ROS in plants maintain a dynamic balance. However, under biotic or abiotic stress, this balance is disrupted, leading to a surge in ROS production and oxidative damage. Compared to normal plant growth, plant tissue culture represents a complex stress that induces the production of large amounts of ROS. Through long-term natural selection, plants have developed an effective reactive oxygen species defense mechanism through enzymatic and non-enzymatic systems.

[0017] Due to the spatiotemporal dynamics of ROS levels induced in plant tissue culture, regulating ROS using upstream and downstream substances can affect the tissue culture effect. Through experimental research, the applicant found that different subculture times have different effects on the regeneration capacity of pine embryonic cells. By classifying different pine embryonic cells according to different subculture times and then using a series of optimized ROS scavengers, the degeneration phenomenon of pine embryonic cells can be effectively improved, providing strong support for the construction of a high-efficiency somatic embryo seedling technology system for pine. [Attached Image Description]

[0018] Figure 1 This is a graph showing the changes in the regeneration capacity of embryonic cells of Pinus massoniana at different subculture times; in the graph, A is the effect of the number of subcultures on the proliferation coefficient; B is the effect of the number of subcultures on the germination rate.

[0019] Figure 2 Ca in pine embryonic cells cultured at different subculture times 2+ Subcellular localization results; In the figure, A represents 10 subcultures, B represents 30 subcultures, the scale bar is 2μm, Cy represents cytoplasm, CW represents cell wall, V represents vacuoles, and IS represents intercellular space.

[0020] Figure 3 These are comparative images of the regeneration effect of embryonic cells from *Pinus massoniana* after different subcultures in the experimental and control groups. In the images, A is a comparative image of the growth effect of embryonic cells after 5 subcultures, with the control group on the left and the experimental group on the right; B is a comparative image of the growth effect of embryonic cells after 10 subcultures, with the control group on the left and the experimental group on the right; C is a comparative image of the growth effect of embryonic cells after 30 subcultures, with the control group on the left and the experimental group on the right.

Detailed Implementation Methods

[0021] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0022] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0023] Example 1:

[0024] This embodiment investigates the effects of adding upstream and downstream substances that regulate ROS production to conventional tissue culture media (e.g., DRC medium) on the regeneration capacity of *Pinus massoniana* embryonic cells at different subculture times. The specific methods are as follows:

[0025] 1. Using immature cones in the split-embryonic stage collected from free-pollinated Masson pine seed orchards as propagation material, embryogenic callus induction was carried out. When the induced transparent, filamentous callus tissue was cultured to 1 cm in size, the embryogenic cells remaining after the embryo was removed were used as the required subculture material.

[0026] 2. The embryonic cells obtained in step 1 were subcultured, and the changes in the regeneration capacity of pine embryonic cells at different subculture times were observed. Through continuous observation of the changes in the regeneration capacity of 14 embryonic cell lines over 2 years of subculture, it was found that the regeneration capacity of embryonic cells varied with different subculture times.

[0027] Specifically, such as Figure 1 As shown, from Figure 1 As can be seen from the AB diagram, the embryogenic cell proliferation capacity (proliferation coefficient) and the ability to regenerate seedlings (germination rate) are both at their maximum after 10 subcultures (140 days), and then gradually decrease. After 30 subcultures (420 days, 1.2 years), the callus tissue almost loses its embryogenicity and has extremely low regeneration capacity.

[0028] Figure 1 The proliferation coefficient refers to the fold increase of embryogenic cells in a single subculture cycle; the germination rate refers to the percentage of mature embryos that can germinate into complete plants after maturation culture. The data shown in the figure are from the observation results of 40 consecutive subcultures of 14 embryogenic cell lines, with a subculture cycle of 14 days / subculture.

[0029] This demonstrates that the growth of *Pinus massoniana* embryonic cells varies with different passage numbers. Therefore, the applicant selected *Pinus massoniana* embryonic cells for culture based on different passage numbers, as detailed below:

[0030] Pine embryonic cells were cultured by adding upstream and downstream substances that regulate ROS production to the conventional tissue culture medium. These substances mainly include glutathione (GSH), calcium ions, and actinomycete ketone (CHX).

[0031] ① Effects of glutathione (GSH) on embryonic cells of Pinus massoniana:

[0032] Glutathione (GSH) is a naturally occurring bioactive secondary metabolite in plants that specifically scavenges reactive oxygen species (ROS). The changes in the regeneration capacity of embryonic cells from *Pinus massoniana* were observed by adding different concentrations of GSH to the culture medium. Figure 1 To investigate the changes in the regeneration capacity of pine embryonic cells at different subculture times, three embryonic cell lines, T1P, T2P, and T6P, were selected as experimental materials. The changes in the regeneration capacity of embryonic cells were observed after the addition of 0–2 mM / L GSH to the culture medium at the 5th, 10th, 15th, 25th, and 30th subcultures.

[0033] The details are shown in Table 1:

[0034] Table 1. Effects of GSH on embryonic cell regeneration capacity of Pinus massoniana at different subculture times.

[0035]

[0036] Note: Different lowercase letters in the table indicate significant differences (p<0.05) between GSH treatments with different generations at the same number of generations.

[0037] The results in Table 1 show that after 5 subcultures, the embryogenic cell proliferation coefficient and germination rate significantly increased with 0.5 mM / L GSH treatment. However, from 10 to 30 subcultures, the embryogenic cell proliferation coefficient and germination rate were highest with 1 mM / L GSH treatment. This indicates that exogenous GSH can significantly enhance the regeneration capacity of pine embryogenic cells, but the concentration of GSH added varies with the subculture time: 0.5 mM / L GSH is added when the number of subcultures is <10, and 1.0 mM / L GSH is added when the number of subcultures is ≥10.

[0038] Adding GSH to the culture medium can increase the proliferation coefficient of embryogenic cells after 5-10 subcultures to 18.3-25.5 and the germination rate to 84.3%-91.2%, with particularly significant improvement. However, for embryogenic cells after 15-30 subcultures, both the proliferation coefficient and germination rate remain at relatively low levels.

[0039] ①Ca 2+ Effects of CHX on embryonic cells of Pinus massoniana:

[0040] The decline in the regenerative capacity of embryonic cells during subculture is essentially due to their degeneration and apoptosis. The fundamental reason GSH plays a role in this application is its scavenging of ROS. From the perspective of the mechanism by which ROS induces plant cell degeneration and apoptosis, firstly, high levels of ROS directly lead to damage to the intracellular membrane system, resulting in metabolic imbalance; secondly, as a signaling molecule, it induces the upstream initiation of MAPKs, Ca... 2 + Abnormal functioning, calcium release from mitochondria, calcium in the cytoplasm 2+ Accumulation leads to the activation of endonucleases, initiating the apoptosis process.

[0041] Specifically, such as Figure 2 As shown, Figure 2 In the diagram, A represents Ca from 10 subcultures and B represents 30 subcultures of *Pinus massoniana* embryonic cells. 2+ Subcellular localization, in the figure, the arrow indicates Ca 2+ Location details, scale bar: 2μm. Cy represents cytoplasm, CW represents cell wall, V represents vacuoles, IS represents intercellular spaces, from... Figure 2 It is evident that after 30 generations, a large amount of Ca accumulated in the cytoplasm of embryonic cells. 2+This indicates that the ROS-induced degeneration of embryonic cells in pine trees is not only due to ROS itself, but also to ROS-induced calcium deficiency in the cells. 2+ Accumulation. To further improve the regeneration capacity of pine embryonic cells in subculture, actinomycete ketone (CHX, a protease inhibitor that can effectively inhibit apoptosis caused by the ROS-activated MAPK pathway) was added to the culture medium and the calcium supply was reduced.

[0042] Table 2. Effects of CHX and calcium supply on embryonic cell regeneration capacity of *Pinus massoniana* at different subculture times.

[0043]

[0044]

[0045] Note: Different lowercase letters in the table indicate significant differences between different calcium supply treatments within the same CHX treatment, while different uppercase letters indicate significant differences between two CHX treatments with the same calcium supply (p<0.05).

[0046] The data shown in the table are average values ​​obtained from observations of multiple embryonic cell lines, with GSH added to the culture medium. When the number of subcultures was 5, the GSH concentration was 0.5 mM / L; when the number of subcultures was 10–30, the GSH concentration was 1.0 mM / L. Calcium supply refers to the amount of Ca(NO3)2·4H2O added to the culture medium, where 556 mg / L Ca(NO3)2·4H2O represents the calcium supply under standard culture conditions.

[0047] As shown in Table 2, the amount of calcium supplied significantly affected the regeneration capacity of embryogenic cells at any number of subcultures. Specifically, at 5 subcultures, the regeneration capacity was optimal under treatment with 278 mg / L Ca(NO3)2·4H2O. However, at 10–30 subcultures, the overall culture effect was better under treatment with 139 mg / L Ca(NO3)2·4H2O. For CHX, the effect was not obvious at 5–25 subcultures, but at 30 subcultures, 10 mg / L CHX significantly improved both the germination and proliferation abilities of embryogenic cells.

[0048] Based on the above experimental observations, the applicant believes the following protocol for the regeneration and culture of pine embryonic cells:

[0049] Table 3. Culture protocols for the regeneration of embryonic cells from *Pinus massoniana* at different passage numbers.

[0050]

[0051] The applicant used the above-mentioned protocol and compared it with a culture medium without added glutathione (GSH), actinomycete ketone (CHX) and high calcium supply (556 mg / L Ca(NO3)2·4H2O).

[0052] Specifically, *Pinus massoniana* embryonic cells with different subculture counts—5 subcultures (70 days), 10 subcultures (140 days), 15 subcultures (210 days), 25 subcultures (350 days), and 30 subcultures (420 days, 1.2 years)—were cultured on the experimental and control culture media, respectively. The control culture medium consisted of the following components: 556 mg / L Ca(NO3)2·4H2O, 400 mg / L ammonium nitrate, 340 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 370 mg / L magnesium sulfate heptahydrate, 85 mg / L calcium chloride dihydrate, 6.2 mg / L boric acid, 22.3 mg / L manganese sulfate hydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.25 mg / L copper sulfate pentahydrate, and 0.83 mg / L... The experimental group's culture medium consisted of: 0.025 mg / L potassium iodide, 0.025 mg / L cobalt chloride hexahydrate, 0.025 mg / L lithium chloride, 0.25 mg / L sodium molybdate dihydrate, 27.8 mg / L ferrous sulfate heptahydrate, 37.3 mg / L disodium EDTA, 1.0 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, 2.0 mg / L glycine, 200 mg / L inositol, 30 g / L sucrose, 3.5 g / L plant gel, 2 g / L 2,4-D, 0.5 g / L 6-BA, 0.5 g / L acid-hydrolyzed casein, and 0.45 g / L glutamine. The experimental group's culture medium was referenced in Table 3, with reduced calcium supply and the addition of GSH and CHX to the control group's medium (DRC medium).

[0053] Embryogenic cells from 5 passages: 0.5 mM / L GSH, 278 mg / L Ca(NO3)2·4H2O, 400 mg / L ammonium nitrate, 340 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 370 mg / L magnesium sulfate heptahydrate, 85 mg / L calcium chloride dihydrate, 6.2 mg / L boric acid, 22.3 mg / L manganese sulfate hydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.25 mg / L copper sulfate pentahydrate, 0.83 mg / L potassium iodide, 0.025 mg / L cobalt chloride hexahydrate 0.025 mg / L lithium chloride, 0.25 mg / L sodium molybdate dihydrate, 27.8 mg / L ferrous sulfate heptahydrate, 37.3 mg / L disodium EDTA, 1.0 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, 2.0 mg / L glycine, 200 mg / L inositol, 30 g / L sucrose, 3.5 g / L plant gel, 2 g / L 2,4-D, 0.5 g / L 6-BA, 0.5 g / L acid-hydrolyzed casein, and 0.45 g / L glutamine;

[0054] Embryogenic cells passaged 10, 15, and 25 times were cultured using the following medium: 1 mM / L GSH, 139 mg / L Ca(NO3)2·4H2O, 400 mg / L ammonium nitrate, 340 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 370 mg / L magnesium sulfate heptahydrate, 85 mg / L calcium chloride dihydrate, 6.2 mg / L boric acid, 22.3 mg / L manganese sulfate hydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.25 mg / L copper sulfate pentahydrate, 0.83 mg / L potassium iodide, and 0.025 mg / L... The following substances were added: cobalt chloride hexahydrate, lithium chloride 0.025 mg / L, sodium molybdate dihydrate 0.25 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, disodium EDTA 37.3 mg / L, thiamine hydrochloride 1.0 mg / L, pyridoxine hydrochloride 0.5 mg / L, nicotinic acid 0.5 mg / L, glycine 2.0 mg / L, inositol 200 mg / L, sucrose 30 g / L, plant gel 3.5 g / L, 2 g / L 2,4-D, 6-BA 0.5 g / L, acid-hydrolyzed casein 0.5 g / L, and glutamine 0.45 g / L.

[0055] Embryogenic cells passaged 30 times: 1 mM / L GSH, 10 mg / L CHX, 139 mg / L Ca(NO3)2·4H2O, 400 mg / L ammonium nitrate, 340 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 370 mg / L magnesium sulfate heptahydrate, 85 mg / L calcium chloride dihydrate, 6.2 mg / L boric acid, 22.3 mg / L manganese sulfate hydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.25 mg / L copper sulfate pentahydrate, 0.83 mg / L potassium iodide, 0.025 mg / L hexahydrate. Cobalt chloride hydrate, lithium chloride 0.025 mg / L, sodium molybdate dihydrate 0.25 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, disodium EDTA 37.3 mg / L, thiamine hydrochloride 1.0 mg / L, pyridoxine hydrochloride 0.5 mg / L, nicotinic acid 0.5 mg / L, glycine 2.0 mg / L, inositol 200 mg / L, sucrose 30 g / L, plant gel 3.5 g / L, 2 g / L, 2,4-D, 6-BA 0.5 g / L, acid-hydrolyzed casein 0.5 g / L, and glutamine 0.45 g / L;

[0056] The results are as follows:

[0057] Table 4. Culture results of regenerated pine embryonic cells after different subcultures under optimized regeneration culture protocols.

[0058]

[0059] Note: Different lowercase letters in the table indicate significant differences (p<0.05) between the experimental and control groups at the same number of generations.

[0060] As shown in Table 4, by using the optimized regeneration technology of the present invention, the proliferation coefficient and germination rate of embryogenic cells in each generation are significantly improved.

[0061] Meanwhile, the growth status of embryonic cells after regeneration in the experimental and control groups was as follows: Figure 3 As shown in the figure, A is a comparison of the growth effect of embryonic cells after 5 subcultures, with the control group on the left and the experimental group on the right; B is a comparison of the growth effect of embryonic cells after 10 subcultures, with the control group on the left and the experimental group on the right; C is a comparison of the growth effect of embryonic cells after 30 subcultures, with the control group on the left and the experimental group on the right. As can be seen from the figure, the embryonic cells in the experimental group are white, have good vitality, and bright color, while the embryonic cells in the control group show significantly increased apoptosis and decline with increasing subculture, and the cells are pale yellow, brown, and severely waterlogged.

[0062] The plant gel used in the examples is G1910-Gelrite produced by Sigma-Aldrich, CAS No. 71010-52-1.

[0063] In summary, this invention explores the effects of adding upstream and downstream substances that regulate ROS production in the culture medium on the regeneration capacity of pine embryonic cells at different subculture times. By utilizing an optimized ROS scavenger in the culture medium, the degeneration of pine embryonic cells is significantly improved, providing strong support for the construction of a high-efficiency somatic embryo seedling technology system for pine.

[0064] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for regenerating embryonic cells of *Pinus massoniana* based on different subculture numbers, characterized in that, The method is as follows: *Pinus massoniana* embryogenic cells are classified according to different subculture numbers, namely, first-generation embryogenic cells, second-generation embryogenic cells, and third-generation embryogenic cells; the first-generation embryogenic cells have been subcultured less than 10 times, the second-generation embryogenic cells have been subcultured 10 to 30 times, and the third-generation embryogenic cells have been subcultured 30 times; glutathione, Ca(NO3)2·4H2O, and / or actinomycete ketone are added to the basal medium according to the different subtypes to obtain the corresponding regeneration medium; the *Pinus massoniana* embryogenic cells are then regenerated using the regeneration medium. The regeneration medium of the first subculture embryonic cells contained 0.5 mM / L glutathione and 278 mg / L Ca(NO3)2·4H2O; The regeneration medium for the second generation of embryonic cells contained 1 mM / L glutathione and 139 mg / L Ca(NO3)2·4H2O. The regeneration medium for the third generation of embryonic cells contained 1 mM / L glutathione, 139 mg / L Ca(NO3)2·4H2O and 10 mg / L actinomycete ketone.

2. The method for regenerating embryonic cells of *Pinus massoniana* according to claim 1, characterized in that, The basal culture medium consists of the following components Composition: DCR medium without Ca(NO3)2·4H2O, 30 g / L sucrose, 3.5 g / L plant gel, 2 g / L 2,4-D, 0.5 g / L 6-BA, 0.5 g / L acid-hydrolyzed casein and 0.45 g / L glutamine.

Images