A culture method for improving rooting efficiency of phoebe delavayi tissue culture seedlings

Abstract

The application discloses a culture method for improving rooting efficiency of Phoebe sheareri tissue culture seedlings, and the rooting efficiency of the Phoebe sheareri tissue culture seedlings is improved through multiple steps of seed disinfection, germination culture, external hormone treatment, active carbon adsorption of secondary metabolites and culture medium optimization, so that the tissue culture efficiency of the Phoebe sheareri is systematically optimized and improved, the survival rate of the Phoebe sheareri tissue culture is improved, and the rooting rate of the Phoebe sheareri is improved from 6.57% to 100%. In addition, the addition of active substances significantly promotes the bolting growth of the Phoebe sheareri, and the bolting rate is increased by about 20% compared with the culture medium without the addition, thereby providing strong support for the subsequent growth of the Phoebe sheareri, especially in the rapid growth of the Phoebe sheareri plant, and providing technical support for commercial application and forestry resource development.

Description

Technical Field

[0001] This invention relates to the field of tissue culture, and specifically to a cultivation method for improving the rooting efficiency of Phoebe zhennan tissue culture seedlings. Background Technology

[0002] Phoebe zhennan, also known as golden nanmu, is a large evergreen tree belonging to the genus Phoebe in the Lauraceae family. It prefers warm and humid climates and is mainly distributed in the subtropical evergreen broad-leaved forests of western Hubei, Sichuan, Chongqing, and Guizhou provinces in my country. It is a national second-class protected tree species endemic to my country. Phoebe zhennan wood is renowned for its unique "golden" luster, making it a major source of this precious timber. Due to its excellent resistance to decay and insects, as well as its properties of being warm in winter and cool in summer, not easily deformed, having a fine grain, and exquisite texture, golden nanmu has been the most ideal and precious building material in traditional Chinese architecture since ancient times, widely used in the construction of palaces, temples, and mausoleums. Phoebe zhennan has long been regarded as a symbol of royalty. Due to over-logging, its existing natural resources are nearing depletion, and it is listed as a second-class protected plant in the "National Key Protected Wild Plants List."

[0003] The propagation techniques for *Phoebe zhennan* mainly include sexual and asexual reproduction. Sexual reproduction relies on the natural acquisition of *Phoebe zhennan* seeds, but seed yield is subject to significant fluctuations between years, and the window for seed maturation and collection is short, meaning seeds only maintain viability for a short period. Furthermore, offspring produced by seed propagation exhibit genetic variation, resulting in unstable seedling traits and difficulty in ensuring the transmission of superior traits. These issues pose challenges to the large-scale propagation of superior *Phoebe zhennan* germplasm. In contrast, asexual propagation techniques such as cuttings and tissue culture can maintain the superior traits of the mother plant and offer rapid propagation, especially tissue culture, which is not limited by season, environment, or site, making it suitable for efficient, large-scale production.

[0004] Current Phoebe zhennan tissue culture techniques still face multiple technical bottlenecks, particularly the inhibition of rooting by secondary metabolite secretion, which severely limits rooting efficiency. Furthermore, the common tissue browning phenomenon during Phoebe zhennan tissue culture, often caused by oxidative stress or nutrient imbalance, further affects normal tissue development and growth. Even when rooting is successful, plant growth remains slow, further prolonging the cultivation cycle and increasing costs. Existing hormone and culture medium optimization protocols are also difficult to standardize, resulting in low experimental reproducibility. These shortcomings significantly increase the complexity and cost of Phoebe zhennan tissue culture techniques.

[0005] To address the shortcomings of Phoebe zhennan, such as low rooting rate, tissue browning, slow growth, and limited technical reserves, this study aims to develop and optimize tissue culture propagation techniques for Phoebe zhennan, providing an innovative solution for rapid and efficient cultivation. Therefore, there is an urgent need for a method to improve the rooting rate and growth efficiency of Phoebe zhennan tissue culture, shorten the propagation cycle, and reduce costs. Summary of the Invention

[0006] In view of this, and addressing the shortcomings of existing technologies, this invention firstly addresses the issue that traditional tissue culture rooting methods typically improve rooting efficiency by adding auxin to the culture medium. However, research has found that short-term external application of auxin after cutting is more effective than adding auxin to the culture medium, significantly improving rooting efficiency. Furthermore, this invention utilizes previously unused active substances, such as astragalus powder, as a culture medium additive, and adds activated carbon to the medium to adsorb secondary metabolites, thereby further improving rooting rate and growth efficiency. This combination of multiple techniques not only significantly improves the rooting rate of *Phoebe zhennan*, but also enhances bolting rate and plant growth, ultimately shortening the propagation cycle and reducing costs.

[0007] The solution of the present invention is as follows:

[0008] 1. A method for improving the rooting efficiency of *Phoebe zhennan* tissue culture seedlings, comprising the following steps:

[0009] (1) Seed disinfection: Take the current year's Phoebe zhennan seeds, wash them and disinfect them with 0.1% mercuric chloride for 20 min;

[0010] (2) Seed germination: The sterilized seeds were placed on a paper-based culture medium for dark germination at a temperature of 23±2℃. The culture medium contained 0.2 g / L activated carbon powder, 30 g / L sucrose, 6 g / L agar, and a modified MS medium with a pH of 5.8.

[0011] (3) Rooting culture: Take the 2-3cm apical bud of the Phoebe zhennan tissue culture seedling obtained in step (2) and insert it into the rooting culture medium. The culture temperature is 23±2℃ and the light is 12h per day. The rooting culture medium contains 200 mg / L activated carbon, 30g / L sucrose, 6 g / L agar, and 1 / 2 MS at pH 5.8.

[0012] Preferably, the present invention also includes bolting culture, wherein the bolting culture involves cutting off the 2-3 cm apical bud of the rooted Phoebe zhennan tissue culture seedling, immersing it in a tissue culture bottle containing 0.5 mg / L indolebutyric acid solution for 15 min, shaking it once every 5 min, and then vertically inserting it into the bolting culture medium for bolting culture. The culture conditions are: culture temperature 23±2℃, light exposure 12h per day.

[0013] In a preferred embodiment of the present invention, in step (3), before inserting the cutting apical bud into the rooting medium, the apical bud is soaked in a hormone solution of 0.5 mg / L IBA or 2 mg / L NAA for 15 min, and shaken once every 5 min.

[0014] Preferably, the bolting culture medium of the present invention is a modified 1 / 2 MS medium containing active substances at pH 5.8, consisting of 200 mg / L activated carbon, 30 g / L sucrose, and 6 g / L agar. The active substances are 50 mg / L of green tea extract, blueberry extract, astragalus extract, or glutathione.

[0015] Preferably, the active substance in this invention is a 50 mg / L Astragalus extract.

[0016] The preferred method for obtaining Phoebe zhennan seeds according to the present invention is as follows: after peeling off the pericarp, wash the seeds three times with detergent, then soak them in a 50 mg / L gibberellin solution for 12 hours, air dry them in a cool and dry place, peel off the seed coat again, and wash them with tap water; after the seed treatment is completed, wash the seeds three times with sterile water in a sterile environment, then rinse them with 75% alcohol for 30 seconds, then wash them three times with sterile water, and finally disinfect them with 0.1% mercuric chloride for 20 minutes.

[0017] The beneficial effects of this invention are as follows: This invention provides a cultivation method for improving the rooting efficiency of *Phoebe zhennan* tissue culture seedlings. While adding auxin to the culture medium can enhance rooting efficiency, research has found that short-term external application of auxin after cutting is more effective than adding auxin directly to the culture medium, significantly improving rooting efficiency. Furthermore, this invention utilizes previously unused active substances, such as astragalus powder, as a culture medium additive, and adds activated carbon to the medium to adsorb secondary metabolites, thereby further improving the rooting rate and growth efficiency. This combination of multiple techniques not only significantly improves the rooting rate of *Phoebe zhennan*, but also results in a more vigorous bolting rate and plant growth, ultimately shortening the propagation cycle and reducing costs. This invention provides a novel solution for the protection and large-scale propagation of this rare tree species, greatly enhancing its feasibility for commercial cultivation. Simultaneously, the optimization and application of this innovative method provides valuable experience for the rapid propagation of other precious tree species, possessing broad application prospects and promotional value. Therefore, this invention is not only of great significance for the protection and utilization of Phoebe zhennan, but also opens up a brand-new technical path for the sustainable development and utilization of precious tree species. Attached Figure Description

[0018] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0019] Figure 1 Examples of different germination culture methods and the results of germination culture for 45 days (A: Germination by Eppendorf tube inoculation; B: Germination by plate inoculation; C: Seedlings 30 days after germination).

[0020] Figure 2 Germination rate of Phoebe zhennan seeds soaked in different concentrations of GA.

[0021] Figure 3 Comparison of rooting effects of Phoebe zhennan tissue culture with and without activated carbon (A: rooting medium without antioxidants; B: rooting medium with PVP; C: rooting medium with activated carbon).

[0022] Figure 4 The bolting effect of adding Astragalus extract to the culture medium. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0024] Improved MS formulation: Improved MS culture medium formulation:

[0025] Potassium nitrate (KNO3): 1900 mg / L; Ammonium nitrate (NH4NO3): 1650 mg / L; Potassium dihydrogen phosphate (KH2PO4): 170 mg / L; Magnesium sulfate (MgSO4·7H2O): 370 mg / L; Calcium chloride (CaCl2·2H2O): 440 mg / L; Potassium iodide (KI): 0.83 mg / L; Boric acid (H3BO3): 6.2 mg / L; Manganese sulfate (MnSO4·4H2O): 22.3 mg / L; Zinc sulfate (ZnSO4·7H2O): 8.6 mg / L; Sodium molybdate (Na2MoO4·2H2O): 0.25 mg / L; Copper sulfate (CuSO4·5H2O): 0.025 mg / L; Potassium sulfate (K2SO4): 1000 mg / L. mg / L, Cobalt chloride (CoCl2・6H2O): 0.025 mg / L, Disodium ethylenediaminetetraacetate (Na2・EDTA): 37.3 mg / L, Ferrous sulfate (FeSO4・7H2O): 27.8 mg / L, Inositol: 100 mg / L, Nicotinic acid (VB3): 0.5 mg / L, Pyridoxine hydrochloride (VB6): 0.5 mg / L, Thiamine hydrochloride (VB1): 0.1 mg / L, Glycine: 2 mg / L.

[0026] Modified 1 / 2 MS: Potassium nitrate (KNO3): 825 mg / L, Ammonium nitrate (NH4NO3): 412.5 mg / L, Potassium dihydrogen phosphate (KH2PO4): 85 mg / L, Magnesium sulfate (MgSO4·7H2O): 92.5 mg / L, Calcium chloride (CaCl2·2H2O): 110 mg / L, Potassium iodide (KI): 0.415 mg / L, Boric acid (H3BO3): 3.1 mg / L, Manganese sulfate (MnSO4·4H2O): 11.15 mg / L, Zinc sulfate (ZnSO4·7H2O): 4.3 mg / L, Sodium molybdate (Na2MoO4·2H2O): 0.125 mg / L, Copper sulfate (CuSO4·5H2O): 0.0125 mg / L, Potassium sulfate (K2SO4): 500 mg / L mg / L, Cobalt chloride (CoCl2・6H2O): 0.0125 mg / L, Disodium ethylenediaminetetraacetate (Na2・EDTA): 18.65 mg / L, Ferrous sulfate (FeSO4・7H2O): 13.9 mg / L, Inositol: 50 mg / L, Nicotinic acid (VB3): 0.25 mg / L, Pyridoxine hydrochloride (VB6): 0.25 mg / L, Thiamine hydrochloride (VB1): 0.05 mg / L, Glycine: 1 mg / L.

[0027] In this embodiment of the invention, the green tea extract was purchased from Shengda Food Additives Co., Ltd.; the blueberry extract was purchased from Wojia Biotechnology Co., Ltd.; the astragalus extract was purchased from Hai'aosi Herbal Essence Workshop; the glutathione was purchased from Beijing Solarbio Technology Co., Ltd.; the vitamin C was purchased from Maclean Biochemical Technology Co., Ltd.; and the citric acid was purchased from Maclean Biochemical Technology Co., Ltd.

[0028] Example 1: Establishment of the Phoebe zhennan tissue culture system

[0029] (1) Establishing a Phoebe zhennan tissue culture system by sterilizing and germinating Phoebe zhennan seeds under tissue culture conditions;

[0030] a. Method for disinfecting Phoebe zhennan seeds: Peel off the pericarp, wash 3 times with detergent, rinse overnight with tap water for 12 hours, air dry, peel off the seed coat, wash with tap water, transfer to a clean bench and wash three times with sterile water (121℃, 20min high-temperature sterilization), rinse with 75% alcohol for 30s, wash 3 times with sterile water, disinfect with disinfectant (mercuric chloride / sodium hypochlorite), wash 6 times with sterile water, and germinate in EP tubes in the dark at a temperature of 23±2℃.

[0031] b. Eppendorf tubes containing culture medium: Modified MS medium + 30 g / L sucrose + 6 g / L agar, pH 5.8, sterilized at 121℃ for 20 min. Place 2 ml of the medium into sterilized 5 ml Eppendorf tubes. Inoculate one seed into each Eppendorf tube at germination time. Figure 1 A).

[0032] The disinfection effects of different disinfection methods were then statistically analyzed, and the results are shown in Table 1.

[0033] Table 1. Disinfection effects of different disinfectants

[0034]

[0035] Disinfection with 0.1% (V / V) mercuric chloride for 20 minutes yields the best results.

[0036] (2) Exploration of germination culture methods:

[0037] The sterilized seeds were placed in Eppendorf tubes (containing culture medium) and paper plates for dark incubation to germinate. Figure 1 B), germination rate was calculated after 30 days ( Figure 1 C).

[0038] The specific method is as follows:

[0039] Eppendorf tubes containing culture medium: modified MS + 0.2 g / L activated carbon powder + 30 g / L sucrose + 6 g / L agar, pH 5.8, sterilized at 121℃ for 20 min. 2 ml of the medium was placed in a sterilized 5 ml Eppendorf tube. One seed was inoculated per Eppendorf tube at germination time. The tubes were cultured in the dark at a temperature of 23±2℃.

[0040] Paper plate germination: Kitchen paper is folded three times and then placed in a plate for high-temperature sterilization. Before germination, 10 ml of sterile water is added to ensure a moist environment. At the time of germination, 9 seeds are inoculated per plate, the plate is sealed with a film to keep it moist, and the plate is placed in a dark environment for germination. The culture temperature is 23±2℃ (note that the seeds are evenly placed during inoculation to avoid large-scale contamination).

[0041] The statistical results are shown in Table 2.

[0042] Table 2. Effects of different germination culture methods

[0043]

[0044] The results showed that the paper plate germination culture method was more conducive to the germination of Phoebe zhennan seeds, with a higher germination rate.

[0045] (3) Treatment of Phoebe zhennan seeds with different concentrations of GA to promote germination:

[0046] To improve rooting efficiency and rooting rate, seeds with the pericarp removed were soaked in gibberellin solutions of 0 mg / L, 50 mg / L, 200 mg / L, 500 mg / L, and 900 mg / L for 24 hours, respectively. After soaking, the seeds were disinfected and then transferred to paper plates for germination at a temperature of 23±2℃. The results are as follows: Figure 2As shown in the figure. The results showed that, under the same conditions, the germination rate of Phoebe zhennan seeds treated with 50 mg / L GA was the highest in tissue culture.

[0047] (4) Adding antioxidants to promote rooting of Phoebe zhennan tissue culture

[0048] To address the challenge of growth inhibition of *Phoebe zhennan* by secondary metabolites under tissue culture conditions, different antioxidants were added to adsorb these secondary metabolites during cultivation. The specific methods are as follows:

[0049] Cut the *Phoebe zhennan* tissue culture seedlings 2-3 cm from the apical bud and insert them vertically into the rooting medium for rooting culture. The rooting culture conditions are: a culture temperature of 23±2℃ and 12 hours of light per day. The rooting medium is prepared as follows:

[0050] No. 1: 1 / 2 MS + 30 g / L sucrose + 6 g / L agar, pH 5.8, sterilized at 121℃ for 20 min;

[0051] No. 2: 1 / 2 MS + 10 mg / L PVP + 30 g / L sucrose + 6 g / L agar, pH 5.8, sterilized at 121℃ for 20 min;

[0052] No. 3: 1 / 2 MS + 200 mg / L activated carbon + 30 g / L sucrose + 6 g / L agar, pH 5.8, sterilized at 121℃ for 20 min.

[0053] Table 3. Rooting rate of Phoebe zhennan tissue culture with added antioxidants

[0054]

[0055] The results showed that adding activated carbon to the culture medium resulted in a rooting rate of 76.92% after 50 days of rooting culture, which was 70% higher than that without the addition of antioxidants.

[0056] (5) Optimization of rooting in tissue culture of Phoebe zhennan

[0057] Cut the *Phoebe zhennan* tissue culture seedlings 2-3 cm from the terminal bud. Immerse them in a tissue culture flask containing a sterile hormone (IBA or NAA) solution for 15 minutes, shaking every 5 minutes. Then, vertically insert them into the rooting medium for rooting culture. The rooting conditions are: temperature 23±2℃, 12 hours of light per day. The sterile hormone solution is prepared as follows: prepare a 1 mg / ml hormone stock solution, filter it through a 20 μm filter for sterilization, and then mix it with sterile water according to the required dosage. The rooting medium is prepared as follows: modified 1 / 2 MS medium + 200 mg / L activated carbon + 30 g / L sucrose + 6 g / L agar, sterilized at pH 5.8, 121℃ for 20 minutes. The rooting rate after 30 days is then calculated, and the results are shown in Table 4.

[0058] Table 4. Rooting rate statistics for auxin external application treatment

[0059]

[0060] The results showed that the rooting efficiency was significantly improved after hormone treatment. After treatment with 0.5 mg / L IBA and then rooting culture in culture medium, the rooting efficiency was better than that under untreated conditions after 50 days of culture in 30 days. Under these conditions, the rooting rate reached 100% after 50 days of culture.

[0061] (6) Adding Astragalus extract to the culture medium promotes bolting growth of Phoebe zhennan tissue culture seedlings.

[0062] To investigate the effects of adding different active substances to the rooting medium of tissue culture subculture on the rooting of *Phoebe zhennan*, *Phoebe zhennan* tissue culture seedlings were cut 2-3 cm from the apical bud. After cutting, they were immersed in a tissue culture bottle containing a sterile 0.5 mg / L indolebutyric acid solution for 15 min, with shaking every 5 min. Then, they were vertically inserted into the rooting medium for rooting culture. The rooting culture conditions were: culture temperature 23±2℃, light 12 h per day. The sterile hormone solution was prepared by: preparing a 1 mg / ml hormone stock solution, filtering it through a 0.22 μm filter for sterilization, and then mixing it with sterile water according to the required amount. The rooting medium was prepared by: modified 1 / 2 MS + 200 mg / L activated carbon + 30 g / L sucrose + 6 g / L agar, sterilized at pH 5.8, 121℃ for 20 min, and then adding the corresponding sterile active substance stock solution. The sterile active substance stock solution was prepared as follows: Weigh 250 mg of active substance powder, add an appropriate amount of ultrapure water, sonicate for 30 min, bring the volume to 10 ml, centrifuge at 12000 rpm for 10 min, and then filter the supernatant through a 0.22 μm filter to remove bacteria, thus obtaining a 25 mg / ml active substance stock solution. Store at 4℃ for later use, and add to the culture medium according to the required dosage. The test results are shown in Table 5.

[0063] Table 5. Effects of adding active substances to the culture medium on bolting rate of Phoebe zhennan after rooting.

[0064]

[0065] The results showed that adding 50 mg / L of filtered and sterilized Astragalus extract to the culture medium was more beneficial to the growth of Phoebe zhennan under tissue culture conditions and increased the bolting rate.

[0066] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A cultivation method for improving the rooting efficiency of *Phoebe zhennan* tissue culture seedlings, characterized in that, Includes the following steps: (1) Seed disinfection: Take the current year's Phoebe zhennan seeds, wash them and disinfect them with 0.1% mercuric chloride for 20 min; (2) Seed germination: The sterilized seeds were placed on a paper-based culture medium for dark germination at a temperature of 23±2℃. The culture medium contained 0.2 g / L activated carbon powder, 30 g / L sucrose, 6 g / L agar, and a modified MS medium with a pH of 5.

8. (3) Rooting culture: Take the 2-3cm apical bud of the Phoebe zhennan tissue culture seedling obtained in step (2) and insert it into the rooting culture medium. The culture temperature is 23±2℃ and the light is 12h per day. The rooting culture medium contains 200 mg / L activated carbon, 30g / L sucrose, 6 g / L agar, and a modified 1 / 2 MS pH 5.

8. Before inserting into the rooting culture medium, soak the cut apical bud in a hormone solution of 0.5 mg / L IBA for 15min and shake it once every 5min. The contents of each component in the modified 1 / 2 MS are as follows: Potassium nitrate: 825 mg / L, Ammonium nitrate: 412.5 mg / L, Potassium dihydrogen phosphate: 85 mg / L, Magnesium sulfate heptahydrate: 92.5 mg / L, Calcium chloride dihydrate: 110 mg / L, Potassium iodide: 0.415 mg / L, Boric acid: 3.1 mg / L, Manganese sulfate tetrahydrate: 11.15 mg / L, Zinc sulfate heptahydrate: 4.3 mg / L, Sodium molybdate dihydrate: 0.125 mg / L, Copper sulfate pentahydrate: 0.0125 mg / L, Potassium sulfate: 500 mg / L, Cobalt chloride hexahydrate: 0.0125 mg / L, Disodium EDTA: 18.65 mg / L, Ferrous sulfate heptahydrate: 13.9 mg / L, Inositol: 50 mg / L, Nicotinic acid: 0.25 mg / L, Pyridoxine hydrochloride: 0.25 mg / L, Thiamine hydrochloride: 0.05 mg / L, glycine: 1 mg / L; The modified MS medium formulation contains the following components: potassium nitrate: 1900 mg / L; ammonium nitrate: 1650 mg / L; potassium dihydrogen phosphate: 170 mg / L; magnesium sulfate heptahydrate: 370 mg / L; calcium chloride dihydrate: 440 mg / L; potassium iodide: 0.83 mg / L; boric acid: 6.2 mg / L; manganese sulfate tetrahydrate: 22.3 mg / L; zinc sulfate heptahydrate: 8.6 mg / L; sodium molybdate dihydrate: 0.25 mg / L; copper sulfate pentahydrate: 0.025 mg / L; potassium sulfate: 1000 mg / L; cobalt chloride hexahydrate: 0.025 mg / L; disodium EDTA: 37.3 mg / L; ferrous sulfate heptahydrate: 27.8 mg / L; inositol: 100 mg / L; nicotinic acid: 0.5 mg / L; pyridoxine hydrochloride: 0.5 mg / L; thiamine hydrochloride: 0.1 mg / L, glycine: 2 mg / L.

2. The cultivation method for improving the rooting efficiency of *Phoebe zhennan* tissue culture seedlings according to claim 1, characterized in that: The method for obtaining Phoebe zhennan seeds is as follows: After peeling off the pericarp, wash the seeds three times with detergent, then soak them in a 50 mg / L gibberellin solution for 12 hours, air dry them in a cool, dry place, peel off the seed coat again, and wash them with tap water. After the seed treatment is completed, wash the seeds three times with sterile water in a sterile environment, then rinse them with 75% alcohol for 30 seconds, then wash them three times with sterile water, and finally disinfect them with 0.1% mercuric chloride for 20 minutes.

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