Culture method for improving rooting efficiency of phoebe zhennan tissue culture seedlings

Abstract

The invention discloses a culture method for improving rooting efficiency of phoebe zhennan tissue culture plants.The method comprises the steps of seed disinfection, germination culture, hormone external application treatment, secondary metabolite adsorption with activated carbon and culture medium optimization, the phoebe zhennan tissue culture efficiency is improved through systematic optimization, the tissue culture survival rate of phoebe zhennan is increased, the rooting rate of phoebe zhennan is increased, and the yield of phoebe zhennan is increased. Compared with the prior art, the method provided by the invention has the advantages that the yield of the phoebe zhennan is increased to 100% from the traditional 6.57%, in addition, the bolting growth of the phoebe zhennan is remarkably promoted by adding active substances, the bolting rate is increased by about 20% compared with that of a culture medium without adding the active substances, powerful support is provided for the subsequent growth of the phoebe zhennan, and particularly, a technical guarantee is provided for commercial application and forestry resource development in the aspect of rapid growth of phoebe zhennan plants.

Description

Technical Field

[0001] The invention relates to the field of tissue culture, and in particular to a culture method for improving the rooting efficiency of Macaoia zhennan tissue culture seedlings. Background Art

[0003] The propagation techniques of Phoebe zhennan mainly include sexual propagation and asexual propagation. Sexual propagation relies on the natural acquisition of Phoebe zhennan seeds, but its seed yield is limited by huge fluctuations between different years, and the window period for seed maturity and collection is short, and the seeds can only remain vigorous in a short period of time. In addition, the offspring produced by seed propagation have genetic variation, and the seedling traits are unstable, making it difficult to ensure the transmission of excellent traits. These problems pose challenges to the large-scale propagation of excellent germplasm of Phoebe zhennan. In contrast, asexual propagation techniques such as cuttings and tissue culture can maintain the excellent traits of the mother plant and have a fast propagation speed, especially tissue culture rapid propagation technology, which is not restricted by season, environment and site, and is suitable for efficient and large-scale production.

[0004] The existing Phoebe zhennan tissue culture technology still faces multiple technical bottlenecks, especially the inhibition of the rooting process by the secretion of secondary metabolites, which seriously limits the rooting efficiency. In addition, the common tissue browning phenomenon in the Phoebe zhennan tissue culture process is mostly caused by oxidative stress or nutritional imbalance, which further affects the normal development and growth of the tissue. Even in the case of successful rooting, the growth rate of the plant is still slow, which further prolongs the cultivation cycle and increases the cost. The existing hormone and culture medium optimization schemes are also difficult to standardize, resulting in low experimental repeatability. These shortcomings have significantly increased the complexity and cost of Phoebe zhennan tissue culture technology.

[0005] In view of the low rooting rate, tissue browning, slow growth, and weak technical reserves of Phoebe zhennan, the tissue propagation technology of Phoebe zhennan is developed and optimized to provide innovative solutions for the rapid and efficient cultivation of Phoebe zhennan. 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, for the deficiencies in the prior art, the present invention first, traditional tissue culture rooting culture usually improves rooting efficiency by adding auxin to the culture medium, but studies have found that short-term external application of auxin treatment after shearing is more effective than adding auxin to the culture medium, greatly improving the rooting efficiency. In addition, the present invention uses active substances that have not been used before, such as astragalus powder as a culture medium additive, and adds activated carbon to the culture medium to adsorb secondary metabolites, thereby further improving the rooting rate and growth efficiency. The combination of these multiple technical means not only significantly improves the rooting rate of Phoebe zhennan, but also the bolting rate and plant growth performance are more vigorous, and ultimately achieves a shortening of the breeding cycle and a reduction in cost.

[0007] The scheme of the present invention is as follows: 1. A method for improving the rooting efficiency of Phoebe zhennan tissue culture seedlings, comprising the following steps: (1) Seed disinfection: Take the seeds of Phoebe zhennan of the current year, wash them and disinfect them with 0.1% mercuric chloride for 20 minutes; (2) Seed germination: The sterilized seeds were placed on a paper-based culture medium for dark germination at a temperature of 23±2°C. 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 2-3 cm apical buds of the Phoebe zhennan tissue culture seedlings germinated in step (2) and insert them into a rooting medium at a temperature of 23±2°C with 12 h of light per day. The rooting medium contains 200 mg / L activated carbon, 30 g / L sucrose, 6 g / L agar, and 1 / 2 MS at pH 5.8.

[0008] Preferably, the present invention further comprises bolting culture, wherein the bolting culture is to cut off the 2-3 cm terminal buds of the rooted Phoebe zhennan tissue culture seedlings, and then soak them in a tissue culture bottle containing 0.5 mg / L indolebutyric acid solution for 15 minutes, shake them once every 5 minutes, and then vertically insert them into the bolting culture medium for bolting culture. The culture conditions are: culture temperature 23±2°C, 12 hours of light per day.

[0009] Preferably, in step (3), the cut apical buds are immersed in a hormone solution of 0.5 mg / L IBA or 2 mg / L NAA for 15 minutes before being inserted into the rooting medium, and are shaken every 5 minutes.

[0010] Preferably, the bolting medium is a modified 1 / 2 MS + 200 mg / L activated carbon + 30 g / L sucrose + 6 g / L agar containing active substances at pH 5.8, and the active substances are 50 mg / L green tea extract, blueberry extract, astragalus extract or glutathione.

[0011] Preferably, the active substance is 50 mg / L of Astragalus extract.

[0012] Preferably, the method for obtaining the Phoebe zhennan seeds is as follows: after peeling the pericarp, wash it with detergent for 3 times, then soak it in a 50 mg / L gibberellin solution for 12 hours, dry it in a cool and dry place, peel off the seed coat and wash it with tap water; after the seed treatment is completed, wash it with sterile water three times in a sterile environment, then rinse it with 75% alcohol for 30 seconds and then wash it with sterile water three times, and finally disinfect it with a volume fraction of 0.1% mercuric chloride for 20 minutes.

[0013] The beneficial effects of the present invention are: a cultivation method for improving the rooting efficiency of Phoebe zhennan tissue culture seedlings of the present invention improves the rooting efficiency by adding auxin to the culture medium, but studies have found that a short-term external application of auxin treatment after shearing is more effective than adding auxin to the culture medium, greatly improving the rooting efficiency. In addition, the present invention uses active substances that have not been used before, such as astragalus powder as a culture medium additive, and adds activated carbon to the culture medium to adsorb secondary metabolites, thereby further improving the rooting rate and growth efficiency. The combination of these multiple technical means not only significantly improves the rooting rate of Phoebe zhennan, but also the bolting rate and plant growth performance are more vigorous, and ultimately achieves a shortening of the breeding cycle and a reduction in cost. The present invention provides a brand-new solution for the protection and large-scale reproduction of the rare tree species Phoebe zhennan, greatly improving its feasibility in commercial planting. At the same time, the optimization and application of this innovative method provides valuable reference experience for the rapid reproduction of other precious tree species, and has broad application prospects and promotion value. Therefore, the present invention is not only of great significance to the protection and utilization of Phoebe zhennan, but also opens up a new technical path for the sustainable development and utilization of precious tree species. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration: Figure 1 Examples of different germination culture methods and the effects of 45 days of germination culture (A: Ep tube inoculation germination; B: plate inoculation germination; C: seedlings 30 days after germination).

[0015] Figure 2 The germination rate of Phoebe zhennan seeds soaked in different concentrations of GA.

[0016] Figure 3 Comparison of the rooting effect of Phoebe zhennan tissue culture with or without the addition of activated carbon (A: no antioxidants were added to the rooting medium; B: PVP was added to the rooting medium; C: activated carbon was added to the rooting medium).

[0017] Figure 4 This is the bolting effect of adding Astragalus extract to the culture medium. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0019] Improved MS medium formula: Improved MS medium formula: Potassium nitrate (KNO3): 1900mg / LAmmonium nitrate (NH4NO3): 1650mg / L, Potassium dihydrogen phosphate (KH2PO4): 170mg / L, Magnesium sulfate (MgSO4・7H2O): 370mg / L, Calcium chloride (CaCl2・2H2O): 440mg / L, Potassium iodide (KI): 0.83mg / L, Boric acid (H3BO3): 6.2mg / L, Manganese sulfate (MnSO4・4H2O): 22.3mg / L, Zinc sulfate (ZnSO4・7H2O): 8.6mg / L, Sodium molybdate (Na2MoO4・2H2O): 0.25mg / L, Copper sulfate (CuSO4・5H2O): 0.025mg / L, Potassium sulfate (K2SO4): 1000 mg / L, cobalt chloride (CoCl2・6H2O): 0.025mg / L, disodium ethylenediaminetetraacetate (Na2・EDTA): 37.3mg / L, ferrous sulfate (FeSO4・7H2O): 27.8mg / L, inositol: 100mg / L, niacin (VB5): 0.5mg / L, pyridoxine hydrochloride (VB6): 0.5mg / L, thiamine hydrochloride (VB1): 0.1mg / L, glycine: 2mg / L.

[0020] Improved 1 / 2 MS: Potassium nitrate (KNO3): 825mg / L, Ammonium nitrate (NH4NO3): 412.5mg / L, Potassium dihydrogen phosphate (KH2PO4): 85mg / L, Magnesium sulfate (MgSO4・7H2O): 92.5mg / L, Calcium chloride (CaCl2・2H2O): 110mg / L, Potassium iodide (KI): 0.415mg / L, Boric acid (H3BO3): 3.1mg / L, Manganese sulfate (MnSO4・4H2O): 11.15mg / L, Zinc sulfate (ZnSO4・7H2O): 4.3mg / L, Sodium molybdate (Na2MoO4・2H2O): 0.125mg / L, Copper sulfate (CuSO4・5H2O): 0.0125mg / L, Potassium sulfate (K2SO4): 500 mg / L, cobalt chloride (CoCl2・6H2O): 0.0125mg / L, disodium ethylenediaminetetraacetate (Na2・EDTA): 18.65mg / L, ferrous sulfate (FeSO4・7H2O): 13.9mg / L, inositol: 50mg / L, niacin (VB5): 0.25mg / L, pyridoxine hydrochloride (VB6): 0.25mg / L, thiamine hydrochloride (VB1): 0.05mg / L, glycine: 1mg / L.

[0021] In the embodiments of the present invention, green tea extract was purchased from Shengda Food Additive Co., Ltd.; blueberry extract was purchased from Wojia Biotechnology Co., Ltd.; astragalus extract was purchased from Haiaosi Herb Essence Shop; glutathione was purchased from Beijing Solebow Technology Co., Ltd.; vitamin C was purchased from MacLean Biochemical Technology Co., Ltd.; citric acid was purchased from MacLean Biochemical Technology Co., Ltd.

[0022] Example 1: Establishment of Phoebe zhennan tissue culture system (1) Establishing a tissue culture system for Phoebe zhennan by sterilizing and germinating Phoebe zhennan seeds under tissue culture conditions; a. Method for disinfecting Phoebe zhennan seeds: peel off the peel, wash with detergent for 3 times, rinse with tap water overnight for 12 hours, dry in the shade, peel off the seed coat, wash with tap water, transfer to the clean bench, wash three times with sterile water (121℃, high temperature sterilization for 20 minutes), rinse with 75% alcohol with shaking for 30 seconds, wash with sterile water for 3 times, disinfect with disinfectant (mercuric chloride / sodium hypochlorite), wash with sterile water for 6 times, and germinate in EP tubes in the dark at a culture temperature of 23±2℃.

[0023] b. Ep tube containing culture medium: modified MS + 30 g / L sucrose + 6 g / L agar, pH 5.8, sterilized at 121°C for 20 min, each 2 ml was placed in a sterilized 5 ml Ep tube, and one seed was inoculated in each Ep tube during germination ( Figure 1 , A).

[0024] Then the disinfection effects of different disinfection methods were statistically analyzed, and the results are shown in Table 1.

[0025] Table 1. Disinfection effect of different disinfectants Disinfectants Contamination rate 0.1% (V / V) mercuric chloride disinfection for 20 minutes 1.59% 0.1% (V / V) mercuric chloride disinfection for 10 minutes 5.39% 2% sodium hypochlorite disinfection for 10 minutes 23.93% The best disinfection effect is achieved by using 0.1% (V / V) mercuric chloride for 20 minutes.

[0026] (2) Research on germination culture methods: The sterilized seeds were placed in Ep tubes (containing culture medium) and paper plates for dark culture for germination ( Figure 1 , B), germination rate was counted after 30 days ( Figure 1 , C).

[0027] The specific method is as follows: Ep tube containing culture medium: improved MS + 0.2 g / L activated carbon powder + 30 g / L sucrose + 6 g / L agar, pH 5.8, high temperature sterilization at 121℃, 20min, each 2 ml is placed in a sterilized 5 ml Ep tube, 1 seed / Ep tube is inoculated during germination, and cultured in a light-proof environment for germination, and the culture temperature is 23±2℃.

[0028] Paper plate germination: Fold the kitchen paper three times and place it on the plate for high temperature sterilization. Add 10 ml of sterile water before germination to ensure a humid environment. Inoculate 9 seeds / plate during germination. Seal the plate to keep it moist. Place it in a light-proof environment for germination. The culture temperature is 23±2℃ (note that the seeds should be placed evenly during inoculation to avoid large amounts of contamination).

[0029] The statistical results are shown in Table 2.

[0030] Table 2. Effects of different germination and culture methods Germination culture method Germination number Number of seeds Germination rate Ep tube germination 175 225 77.78% Paper Plate Germination 209 225 92.89% The results showed that the germination culture method of paper plate germination was more conducive to the germination of Phoebe zhennan seeds and had a higher germination rate.

[0031] (3) Soaking Phoebe zhennan seeds in different concentrations of GA to promote germination: In order to improve the rooting efficiency and rooting rate, the seeds with peel removed were soaked in 0 mg / L, 50 mg / L, 200 mg / L, 500 mg / L, and 900 mg / L gibberellin solutions for 24 hours, disinfected after soaking, and then transferred to paper plates for germination. The culture temperature was 23±2℃. The results are as follows Figure 2 The results showed that under the same conditions, the germination rate of Phoebe zhennan seeds treated with 50 mg / L GA was the highest under tissue culture conditions.

[0032] (4) Adding antioxidants to promote rooting of Phoebe zhennan tissue culture In order to solve the problem that the growth of Phoebe zhennan is inhibited by secondary metabolites under tissue culture conditions, different antioxidants were added to absorb secondary metabolites for cultivation. The specific method is as follows: The Phoebe zhennan tissue culture seedlings were cut 2-3 cm away from the terminal buds and vertically inserted into the rooting medium for rooting culture. The rooting culture conditions were: culture temperature 23±2°C, light exposure 12h per day, and the rooting medium was prepared as follows: No. 1: 1 / 2 MS + 30 g / L sucrose + 6 g / L agar, pH 5.8, high temperature sterilization at 121°C for 20 min; No. 2: 1 / 2 MS + 10 mg / L PVP + 30 g / L sucrose + 6 g / L agar, pH 5.8, high temperature sterilization at 121°C for 20 min; No. 3: 1 / 2 MS + 200 mg / L activated carbon + 30 g / L sucrose + 6 g / L agar, pH 5.8, high temperature sterilization at 121℃ for 20 min.

[0033] Table 3. Rooting rate of Phoebe zhennan tissue culture with antioxidants added to the culture medium Content of each component of the culture medium Rooting rate 1 / 2 MS + 30g / L sucrose 6.57% 1 / 2 MS + 10 mg / L PVP + 30g / L sucrose 16．00% 1 / 2 MS + 200 mg / L activated carbon + 30g / L sucrose 76.92% The results showed that when activated carbon was added to the culture medium, the rooting rate reached 76.92% after 50 days of rooting culture, which was 70% higher than that without adding antioxidants.

[0034] (5) Optimization of rooting of Phoebe zhennan in tissue culture The Phoebe zhennan tissue culture seedlings were cut 2-3 cm away from the terminal buds, and then placed in a tissue culture bottle containing sterile hormone (IBA or NAA) solution for immersion for 15 minutes, shaken once every 5 minutes, and then vertically inserted into the rooting medium for rooting culture. The conditions for rooting culture are: culture temperature 23±2℃, light 12h per day, the sterile hormone solution preparation method is: prepare 1mg / ml hormone mother solution, filter and sterilize with a 20μm filter head, and then mix with sterile water according to the required amount; the rooting medium preparation method is: modified 1 / 2 MS +200 mg / L activated carbon +30g / L sucrose +6g / L agar, pH5.8, 121℃, 20min high temperature sterilization. Then the rooting rate 30d after treatment was counted, and the results are shown in Table 4.

[0035] Table 4. Rooting rate statistics of auxin application treatment Processing Rooting rate 30 days after treatment Unprocessed 40.74% 0.5 mg / L IBA 83.33% 2 mg / L NAA 66.67% The results showed that the rooting efficiency was significantly improved after hormone treatment. After applying 0.5 mg / L IBA treatment and then inserting into the culture medium for rooting culture, culturing for 30 days could achieve better results than culturing for 50 days under untreated conditions. Under this condition, the rooting rate reached 100% after 50 days of culture.

[0036] (6) Adding Astragalus extract to the culture medium promotes the bolting growth of Phoebe zhennan tissue culture seedlings In order to study the effect of adding different active substances to the tissue culture subculture rooting medium on the rooting of Phoebe zhennan, the Phoebe zhennan tissue culture seedlings were cut 2-3 cm away from the terminal bud, and then placed in a tissue culture bottle containing a sterile 0.5 mg / L indolebutyric acid solution for 15 minutes, shaken once every 5 minutes, and then vertically inserted into the rooting medium for rooting culture. The conditions for rooting culture are: culture temperature 23±2℃, light 12h per day, the preparation method of the sterile hormone solution is: prepare 1mg / ml hormone stock solution, filter and sterilize with a 0.22um filter head, and then mix and shake the required amount with sterile water; the preparation method of the rooting medium is: modified 1 / 2 MS + 200 mg / L activated carbon + 30g / L sucrose + 6 g / L agar, pH5.8, 121℃, 20min high temperature sterilization, and then add the corresponding sterile active substance stock solution. The method for preparing the sterile active substance mother solution is as follows: weigh 250 mg of active substance powder, add appropriate amount of ultrapure water and ultrasonically dissolve for 30 minutes, make the volume to 10 ml and centrifuge at 12000 rpm for 10 minutes, take the supernatant after centrifugation and filter and sterilize with a 0.22 um filter to obtain a 25 mg / ml active substance mother solution, store it in a 4°C refrigerator for use, and add it to the culture medium according to the required amount when using it. The test results are shown in Table 5.

[0037] Table 5. Effects of adding active substances to the culture medium on the bolting rate of Phoebe zhennan after rooting Add active ingredients Bolting rate No Addition 38.89% Green Tea Extract 26.67% Blueberry Extract 61.11% Astragalus Extract 66.67% Glutathione 61.11% Vitamin C 38.10% Citric Acid 42.86% The results showed that adding 50 mg / L of filtered and sterilized Astragalus extract to the culture medium was more conducive to the growth of Phoebe zhennan under tissue culture conditions and increased the bolting rate.

[0038] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A method for improving the rooting efficiency of Phoebe zhennan tissue culture seedlings, characterized in that: The steps include: (1) Seed disinfection: Take the seeds of Phoebe zhennan of the current year, wash them and disinfect them with 0.1% mercuric chloride for 20 minutes; (2) Seed germination: The sterilized seeds were placed on a paper-based culture medium for dark germination at a temperature of 23±2°C. 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 2-3 cm apical buds of the Phoebe zhennan tissue culture seedlings germinated in step (2) and insert them into a rooting medium at a temperature of 23±2°C with 12 h of light per day. The rooting medium contains 200 mg / L activated carbon, 30 g / L sucrose, 6 g / L agar, and 1 / 2 MS at pH 5.

8.

2. The method for improving the rooting efficiency of Phoebe zhennan tissue culture seedlings according to claim 1, characterized in that: The method also includes bolting culture, wherein the 2-3 cm top buds of the rooted Phoebe zhennan tissue culture seedlings are cut off, then immersed in a tissue culture bottle containing 0.5 mg / L indolebutyric acid solution for 15 minutes, shaken once every 5 minutes, and then vertically inserted into the bolting culture medium for bolting culture. The culture conditions are: culture temperature 23±2°C, 12 hours of light per day.

3. The method for improving the rooting efficiency of Phoebe zhennan tissue culture seedlings according to claim 1, characterized in that: In step (3), before inserting into the rooting medium, the cut apical buds were immersed in a hormone solution of 0.5 mg / L IBA or 2 mg / L NAA for 15 minutes, with shaking every 5 minutes.

4. The method for improving the rooting efficiency of Phoebe zhennan tissue culture seedlings according to claim 2, characterized in that: The bolting medium is pH 5.8, modified 1 / 2 MS + 200 mg / L activated carbon + 30 g / L sucrose + 6 g / L agar containing active substances, and the active substances are 50 mg / L green tea extract, blueberry extract, astragalus extract or glutathione.

5. The method for improving the rooting efficiency of Phoebe zhennan tissue culture seedlings according to claim 4, characterized in that: The active substance is 50 mg / L of astragalus extract.

6. The method for improving the rooting efficiency of Phoebe zhennan tissue culture seedlings according to claim 1, characterized in that: The method for obtaining the Phoebe zhennan seeds is as follows: after peeling the pericarp, wash it with detergent for 3 times, then soak it in a 50 mg / L gibberellin solution for 12 hours, dry it in a cool and dry place, peel the seed coat and wash it with tap water; after the seed treatment is completed, wash it with sterile water for 3 times in a sterile environment, then rinse it with 75% alcohol for 30 seconds and then wash it with sterile water for 3 times, and finally disinfect it with 0.1% mercuric chloride by volume for 20 minutes.

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