Application of flavonoids in promoting embryogenic maintenance of korean pine callus and somatic embryogenesis of korean pine

Abstract

The application discloses application of flavonoids in promoting embryogenic maintenance of Pinus koraiensis callus and somatic embryogenesis of Pinus koraiensis and belongs to the technical field of plant tissue regeneration. The application provides application of flavonoids in promoting embryogenic maintenance of Pinus koraiensis callus, somatic embryogenesis of Pinus koraiensis and reduction of active oxygen, wherein the flavonoids are quercetin, naringenin or catechin. The method improves the genetic improvement efficiency of Pinus koraiensis.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue regeneration technology, specifically involving the application of flavonoids in promoting the maintenance of embryogenicity of callus tissue and embryogenesis in Korean pine. Background Technology

[0002] Korean pine is a precious native tree species in my country, and the dominant species of Korean pine broad-leaved forests in temperate humid zonal climax communities, possessing significant ecological, economic, and social value. Artificial cultivation of Korean pine began relatively late, and currently, artificial reserve resources are severely insufficient, while the superior germplasm resources available for resource expansion are very limited. Currently, the main propagation methods for Korean pine include seed propagation and grafting. However, due to the inherent biological characteristics of Korean pine, seed propagation involves slow seedling growth and a long reproductive cycle. In natural forests, it takes approximately 80 years for Korean pine to flower and bear fruit, completing a full life cycle, while in plantations or seed orchards, it takes 15-25 years. Therefore, genetic improvement of Korean pine using conventional breeding methods requires enormous time and resources. Furthermore, problems such as large genetic variations and unstable trait inheritance in hybrid offspring may occur. Regarding grafting propagation, although current grafting techniques are relatively mature, grafting requires the collection of scions and the cultivation of rootstocks, demanding high technical skills and resulting in high labor costs. Simultaneously, the grafting propagation coefficient is insufficient to meet the needs of large-scale afforestation. Therefore, the establishment of an efficient somatic embryogenesis technology system is of great significance for improving the genetic improvement efficiency of Korean pine and solving the problem of scarcity of high-quality germplasm.

[0003] Somatic embryogenesis is one of the important means to achieve efficient and large-scale propagation of superior tree varieties and preservation of excellent traits in forest tree reproduction and genetic breeding. In recent years, during the development of the somatic embryogenesis system of Korean pine, it has been found that the differentiation ability of embryogenic callus induced by immature zygotes decreases rapidly with the increase of subculture. After 12 months of proliferation culture, the callus can basically not differentiate into mature somatic embryos. Similar phenomena are also common in other tree species, which will become one of the key technical bottlenecks for the promotion and application of the somatic embryogenesis pathway in Korean pine. Summary of the Invention

[0004] The purpose of this invention is to improve the efficiency of somatic embryogenesis in red pine callus tissue.

[0005] This invention provides the application of flavonoids in promoting the embryogenicity of callus tissue in Korean pine, the embryogenesis of Korean pine, and reducing reactive oxygen species.

[0006] Further specifying, the flavonoids are quercetin, naringenin, or catechin.

[0007] This invention provides a method for promoting the maintenance of embryogenicity of Korean pine callus, the development of Korean pine seroembryo, and the reduction of reactive oxygen species. The specific steps of the method are as follows: Korean pine callus is added to a proliferation medium containing flavonoids, cultured in the dark, and then cultured to mature, thereby inducing Korean pine seroembryo.

[0008] Further specifying, the flavonoids are quercetin, naringenin, or catechin.

[0009] Furthermore, the concentration of flavonoids is limited to no more than 20 μM.

[0010] Further specify the conditions for dark culture: 25°C for 9-12 days.

[0011] Further specifying, the formulation of the proliferation medium is as follows: based on mLV, supplemented with 2 mg·L⁻¹ -1 2,4-D, 0.5 mg·L -1 6-BA, 25g·L -1 sucrose, 0.5 g·L -1 Acid-hydrolyzed casein and 0.5 g·L -1 L-glutamine, 4 g·L -1 The gelling gum.

[0012] Further, after dark culture, the red pine callus tissue was added to the suspension culture medium to suspend and disperse the cells. The dispersed cells were then filtered through a Buchner funnel and placed in the maturation culture medium for maturation culture. The suspended cells were cultured in the maturation culture medium at 25°C in the dark for 60 days.

[0013] Further specifying the composition of the suspension culture medium: based on mLV, with the addition of 25 g·L⁻¹ -1 sucrose, 0.5 g·L -1 Acid-hydrolyzed casein, 0.5 g·L -1 L-glutamine.

[0014] Further specifying the composition of the maturation medium: based on mLV, with the addition of 8 g·L⁻¹ -1 gellan gum, 68 g·L -1 sucrose, 1 g·L -1 Activated carbon, 0.5 g·L -1 Acid-hydrolyzed casein, 0.5 g·L -1 Glutamine and 21 mg·L -1 ABA.

[0015] Beneficial Effects: This invention utilizes exogenous flavonoids (quercetin, naringenin, and catechins) to treat Korean pine callus with reduced embryogenicity during the proliferation stage. It was found that exogenous application of flavonoids significantly improves the somatic embryogenesis efficiency of Korean pine callus. Through treatment with different concentrations of flavonoids, we found that the highest somatic embryogenesis efficiency was achieved under the treatment conditions of 20 μM quercetin, naringenin, and catechins, increasing by 880%, 1450%, and 1110%, respectively, compared to no flavonoid treatment. Simultaneously, after flavonoid application, the reactive oxygen species (ROS) in the callus tissue were significantly reduced, specifically reflected in histochemical staining, fluorescence detection, and physiological assays. Attached Figure Description

[0016] Figure 1 Figure showing the effect of exogenous flavonoid treatment on embryogenesis rate in Korean pine.

[0017] Figure 2 Figure showing the effect of exogenous flavonoids on cell morphology of Pine callus tissue;

[0018] Figure 3 The figure shows the ROS level measurement results in callus tissue after flavonoid treatment. Detailed Implementation

[0019] DCF Detection: Intracellular ROS levels were assessed using the ROS-specific fluorescent probe H2DCF. H2DCF was dissolved in DMSO to prepare a 10 mM stock solution. 5 μL of H2DCF was added to 5 mL of 1×PBS buffer to obtain a final staining solution concentration of 10 μM. The callus tissue and staining solution were incubated in the dark at room temperature for 5–15 min (placed in a 6-well plate and wrapped with aluminum foil). The tissue was then washed with 1×PBS for 5 minutes (excess liquid was aspirated with a pipette), repeated 3 times. Images were immediately captured using a fluorescence stereomicroscope.

[0020] DAB and NBT detection: DAB is reddish-brown; NBT is dark blue.

[0021] Dissolve DAB powder in Tris-HCl (pH=3.8) to prepare a 1 mg / mL solution. Immerse the callus tissue in the dark for 5–10 min. H2O2 will stain it reddish-brown. Take a picture using a stereomicroscope.

[0022] Dissolve NBT powder in 1xPBS (pH=7.5) to prepare a 2 mg / mL solution. Immerse the callus tissue in the dark for 2–5 min. - It can be stained dark blue and photographed using a stereomicroscope.

[0023] Example 1. A method for promoting the maintenance of embryogenicity of Korean pine callus and the development of Korean pine seroembryo using quercetin.

[0024] 1. Prepare callus proliferation medium, and add 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM quercetin to the medium, respectively. The proliferation subculture medium formula is: mLV plus 2 mg·L⁻¹ -1 2,4-D, 0.5 mg·L -1 6-BA, 25g·L -1 Sucrose, 0.5 g·L -1 Acid-hydrolyzed casein, 0.5 g·L -1 L-glutamine and 4 g·L -1 Gellan gel. Adjust the pH to 5.8. Note: Add L-glutamine only after autoclaving at 121°C.

[0025] 2. Pour the culture medium into 90mm petri dishes and allow it to cool and solidify. After about 1 hour, transfer the red pine embryogenic callus tissue that needs to be subcultured to fresh proliferation medium using sterilized forceps. There are 3 dishes for each treatment, and each dish contains 5 clumps of 0.1g fresh callus tissue. Place the petri dishes in the dark at 25℃.

[0026] 3. After 12 days of proliferation, 0.35g of the proliferated callus tissue was placed in a 50mL centrifuge tube containing 15mL of suspension culture medium for resuscitation and dispersion (suspension culture medium: based on mLV, supplemented with 25g·L⁻¹). -1 sucrose, 0.5 g·L -1 Acid-hydrolyzed casein and 0.5 g·L -1 (L-glutamine), gently shake the conical flask for 10 minutes.

[0027] 4. After the cells disperse, use a pipette tip to draw 3 mL of the suspension and transfer it onto filter paper. Place the sterile filter paper on a Buchner funnel and perform vacuum filtration until no water drips off the filter paper (1 min).

[0028] 5. Use tweezers to transfer the filter paper into the maturation culture medium. The formula for the maturation culture medium is: mLV + 8 g·L⁻¹ -1 Gellan gum + 68g·L -1 Sucrose + 1g·L -1 Activated carbon + 0.5 g·L -1 Casein was acid-hydrolyzed to pH 5.8, then autoclaved and placed in a clean bench. The temperature was lowered to approximately 45°C, and then 0.5 g / L of filtered and sterilized [material / solvent] was added. -1 Glutamine and 21 mg·L -1 ABA.

[0029] 6. Place 5 culture dishes containing callus in the dark at 25°C for maturation culture.

[0030] 7. After 60 days of maturation culture, observe the somatic embryos under a stereomicroscope (SZX-ILLB2-200, Olympus Corporation, Tokyo, Japan) and count the number of somatic embryos. Number of somatic embryos (units / g) = Number of somatic embryos / Fresh weight of embryogenic callus.

[0031] Example 2. A method for promoting the maintenance of embryogenicity of Korean pine callus and the development of Korean pine seroembryo using naringenin.

[0032] 1. Prepare callus proliferation medium, and add 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM naringenin to the medium, respectively. The proliferation subculture medium formula is: mLV plus 2 mg·L⁻¹ -1 2,4-D, 0.5 mg·L -1 6-BA, 25g·L -1 Sucrose, 0.5 g·L -1 Acid-hydrolyzed casein, 0.5 g·L -1 L-glutamine and 4 g·L -1 Gellan gel. Adjust the pH to 5.8. Note: Add L-glutamine only after autoclaving at 121°C.

[0033] 2. Pour the culture medium into 90mm petri dishes and allow it to cool and solidify. After about 1 hour, transfer the red pine embryogenic callus tissue that needs to be subcultured to fresh proliferation medium using sterilized forceps. There are 3 dishes for each treatment, and each dish contains 5 clumps of 0.1g fresh callus tissue. Place the petri dishes in the dark at 25℃.

[0034] 3. After 12 days of proliferation, 0.35g of the proliferated callus tissue was placed in a 50mL centrifuge tube containing 15mL of suspension culture medium for resuscitation and dispersion (suspension culture medium: based on mLV, supplemented with 25g·L⁻¹). -1 sucrose, 0.5 g·L -1 Acid-hydrolyzed casein and 0.5 g·L -1 (L-glutamine), gently shake the conical flask for 10 minutes.

[0035] 4. After the cells disperse, use a pipette tip to draw 3 mL of the suspension and transfer it onto filter paper. Place the sterile filter paper on a Buchner funnel and perform vacuum filtration until no water drips off the filter paper (1 min).

[0036] 5. Use tweezers to transfer the filter paper into the maturation culture medium. The formula for the maturation culture medium is: mLV + 8 g·L⁻¹ -1 Gellan gum + 68g·L -1 sucrose + 1g·L -1 Activated carbon + 0.5 g·L -1Casein was acid-hydrolyzed to pH 5.8, then autoclaved and placed in a clean bench. The temperature was lowered to approximately 45°C, and then 0.5 g / L of filtered and sterilized [material / solvent] was added. -1 Glutamine and 21 mg·L -1 ABA.

[0037] 6. Place 5 culture dishes containing callus in the dark at 25°C for maturation culture.

[0038] 7. After 60 days of maturation culture, observe the somatic embryos under a stereomicroscope (SZX-ILLB2-200, Olympus Corporation, Tokyo, Japan) and count the number of somatic embryos. Number of somatic embryos (units / g) = Number of somatic embryos / Fresh weight of embryogenic callus.

[0039] Example 3. A method for promoting the maintenance of embryogenicity of Korean pine callus and the development of Korean pine seroembryo using catechins.

[0040] 1. Prepare callus proliferation medium, and add 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM catechins to the medium, respectively. The proliferation subculture medium formula is: mLV plus 2 mg·L⁻¹ -1 2,4-D, 0.5 mg·L -1 6-BA, 25g·L -1 Sucrose, 0.5 g·L -1 Acid-hydrolyzed casein, 0.5 g·L -1 L-glutamine and 4 g·L -1 Gellan gel. Adjust the pH to 5.8. Note: Add L-glutamine only after autoclaving at 121°C.

[0041] 2. Pour the culture medium into 90mm petri dishes and allow it to cool and solidify. After about 1 hour, transfer the red pine embryogenic callus tissue that needs to be subcultured to fresh proliferation medium using sterilized forceps. There are 3 dishes for each treatment, and each dish contains 5 clumps of 0.1g fresh callus tissue. Place the petri dishes in the dark at 25℃.

[0042] 3. After 12 days of proliferation, 0.35g of the proliferated callus tissue was placed in a 50mL centrifuge tube containing 15mL of suspension culture medium for resuscitation and dispersion (suspension culture medium: based on mLV, supplemented with 25g·L⁻¹). -1 sucrose, 0.5 g·L -1 Acid-hydrolyzed casein and 0.5 g·L -1 (L-glutamine), gently shake the conical flask for 10 minutes.

[0043] 4. After the cells disperse, use a pipette tip to draw 3 mL of the suspension and transfer it onto filter paper. Place the sterile filter paper on a Buchner funnel and perform vacuum filtration until no water drips off the filter paper (1 min).

[0044] 5. Use tweezers to transfer the filter paper into the maturation culture medium. The formula for the maturation culture medium is: mLV + 8 g·L⁻¹ -1 Gellan gum + 68g·L -1 sucrose + 1g·L -1 Activated carbon + 0.5 g·L -1 Casein was acid-hydrolyzed to pH 5.8, then autoclaved and placed in a clean bench. The temperature was lowered to approximately 45°C, and then 0.5 g / L of filtered and sterilized [material / solvent] was added. -1 Glutamine and 21 mg·L -1 ABA.

[0045] 6. Place 5 culture dishes containing callus in the dark at 25°C for maturation culture.

[0046] 7. After 60 days of maturation culture, observe the somatic embryos under a stereomicroscope (SZX-ILLB2-200, Olympus Corporation, Tokyo, Japan) and count the number of somatic embryos. Number of somatic embryos (units / g) = Number of somatic embryos / Fresh weight of embryogenic callus.

[0047] Results: Sixty days after somatic embryogenesis, the number of somatic embryos in the callus tissue supplemented with flavonoids was counted, and the somatic embryogenesis efficiency was calculated. Figure 1 a). From Figure 1 As shown in b, the number of somatic embryos significantly increased after the addition of flavonoids. Furthermore, cell morphology analysis of callus 7 days after flavonoid addition revealed an increase in the number of early somatic embryos in the callus tissue. Figure 2 Simultaneously, physiological and biochemical methods were used to detect the reactive oxygen species (ROS) content in flavonoid-treated callus tissue. H2DCF, NBT, and DAB were used to detect the ROS levels and singlet oxygen (O2) in the tissue, respectively. - The content of reactive oxygen species was found to be significantly reduced under flavonoid treatment, along with the content of hydrogen peroxide (H2O2). Figure 3 This indicates that flavonoids promote somatic embryogenesis efficiency by reducing reactive oxygen species in callus tissue and increasing the number of early somatic embryos in callus tissue.

[0048] Treatment with different concentrations of flavonoids revealed that the highest somatic embryogenesis efficiency was achieved under treatment with 20 μM quercetin, naringenin, and catechin, increasing by 880%, 1450%, and 1110%, respectively, compared to the control group (without flavonoid treatment). Simultaneously, flavonoid application significantly reduced reactive oxygen species (ROS) in the callus tissue, as evidenced by improvements in histochemical staining, fluorescence detection, and physiological assays. The results are shown in Table 1.

[0049] Table 1. Effects of different flavonoid treatments on the number of embryos in embryogenic callus from Pine pine.

[0050]

[0051] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05).

Claims

1. The application of flavonoids in promoting embryogenesis and reducing reactive oxygen species in Korean pine, characterized in that, The flavonoids were quercetin, naringenin, or catechin; the concentration of the flavonoids was 20 μM.

2. A method for promoting embryogenesis in Korean pine and reducing reactive oxygen species, characterized in that, The specific steps of the method are as follows: Korean pine callus tissue is added to a proliferation medium containing flavonoids, cultured in the dark, and then subjected to maturation culture to induce Korean pine embryogenesis. The proliferation medium formula is as follows: based on mLV, supplemented with 2 mg·L⁻¹ -1 2,4-D, 0.5 mg·L -1 6-BA, 25 g·L -1 Sucrose, 0.5 g·L -1 Acid-hydrolyzed casein, 0.5 g·L -1 L-glutamine, 4 g·L -1 The mixture contained gellan gum and 20 μM quercetin, naringenin, or catechin; the composition of the maturation medium used for maturation culture was: based on mLV, supplemented with 8 g·L⁻¹ -1 gellan gum, 68 g·L -1 sucrose, 1 g·L -1 Activated carbon, 0.5 g·L -1 Acid-hydrolyzed casein, 0.5 g·L -1 Glutamine and 21 mg·L -1 ABA.

3. The method according to claim 2, characterized in that, The conditions for dark culture are 25℃ for 9-12 days.

4. The method according to claim 2, characterized in that, After dark culture, the red pine callus tissue was added to the suspension culture medium to suspend and disperse the cells. The dispersed cells were then filtered through a Buchner funnel and placed in the maturation culture medium for maturation culture. The suspension cells were cultured in the maturation culture medium at 25°C in the dark for 60 days.

5. The method according to claim 4, characterized in that, Composition of suspension medium: based on mLV, with 25 g·L⁻¹ added. -1 Sucrose, 0.5 g·L -1 Acid-hydrolyzed casein and 0.5 g·L -1 L-glutamine.

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