A method for meristem virus-free tissue culture of top buds of flowered cattle apple rootstock

Abstract

This invention discloses a method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock, belonging to the field of plant tissue culture technology. The method includes: selecting terminal buds of *Prunus armeniaca* rootstock for pretreatment, sterilizing them with ethanol and mercuric chloride, and then immersing them in a virus-free pretreatment solution containing ribavirin and triazole nucleoside for virus removal; inoculating the virus-free terminal buds into WPM-based differentiation induction medium to obtain clustered buds; culturing the clustered buds in 1 / 2 MS-based proliferation subculture medium to obtain proliferating buds; inoculating the proliferating buds into 1 / 3 MS-based rooting medium to induce rooting; and finally, after hardening off and transplanting into a vermiculite-garden soil-humus substrate for acclimatization. This invention optimizes the virus removal and tissue culture system based on the characteristics of *Prunus armeniaca* rootstock, achieving a virus removal rate ≥95%, a terminal bud differentiation survival rate ≥85%, a rooting rate ≥90%, and a transplant survival rate ≥88%. This enables rapid and large-scale propagation of virus-free *Prunus armeniaca* rootstock seedlings, solving the problem of low propagation efficiency in traditional methods and providing high-quality rootstock support for the *Prunus armeniaca* industry.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for virus-free tissue culture using the terminal buds of *Prunus armeniaca* rootstock. This method is suitable for the virus-free and rapid propagation of *Prunus armeniaca* rootstock and can significantly improve the germplasm purity and propagation efficiency of *Prunus armeniaca* rootstock. Background Technology

[0002] Huaniu apple is a famous apple variety in my country, known for its excellent quality and unique taste, and enjoys high economic value in the market. Rootstock, as the foundation of apple tree growth, directly affects the growth vigor, stress resistance, fruiting period, and fruit quality of Huaniu apples. However, traditional Huaniu apple rootstock propagation methods, such as grafting and cutting, are prone to virus accumulation, leading to weakened rootstock growth and reduced resistance to pests and diseases, thus affecting the yield and quality of Huaniu apples. Furthermore, conventional tissue culture methods suffer from poor virus elimination, low apical bud differentiation rate, unstable rooting rate, and low transplant survival rate, making it difficult to meet the large-scale demand of the Huaniu apple industry for high-quality virus-free rootstocks.

[0003] Currently, existing apple rootstock tissue culture techniques are mostly designed for common apple rootstocks (such as M9 and common dwarfing rootstocks), without being optimized for the physiological characteristics of Huaniu apple rootstocks. This makes it impossible to balance virus elimination effectiveness with tissue culture propagation efficiency. Therefore, developing a rapid tissue culture propagation method specifically for virus elimination from the terminal buds of Huaniu apple rootstocks is of great significance for improving the quality of Huaniu apple rootstocks and promoting the standardized development of the Huaniu apple industry. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock. This method optimizes key steps such as terminal bud disinfection, virus-free treatment, differentiation culture, proliferation culture, rooting culture, and transplanting acclimatization to achieve efficient virus-free and rapid propagation of terminal buds of *Prunus armeniaca* rootstock. The virus-free rate can reach over 95%, the terminal bud differentiation survival rate is over 85%, the rooting rate is over 90%, and the transplanting survival rate is over 88%.

[0005] To achieve the above objectives, this invention provides a method for virus-free tissue culture using the terminal buds of *Prunus armeniaca* rootstock, specifically comprising the following steps:

[0006] S1 Explant Selection and Pretreatment

[0007] Select the terminal buds at the tips of the current year's new shoots of healthy, disease-free apple rootstock plants, preferably with a length of 1.5-2.0cm. Remove the outer old leaves of the terminal bud and retain 2-3 inner young leaves. Rinse with running water for 30 minutes to remove surface impurities, then rinse three times with sterile water and place in a clean bench for later use.

[0008] S2 apical bud disinfection and detoxification treatment

[0009] 1. Disinfection steps: Place the pretreated apical bud in a sterile conical flask, add 75% ethanol solution for 30 seconds, rinse 3 times with sterile water; then add 0.1% mercuric chloride solution, add 1-2 drops of Tween-80, shake for 6 minutes, rinse 5 times with sterile water, and finally blot dry the surface moisture of the apical bud with sterile filter paper.

[0010] 2. Detoxification treatment: The disinfected apical buds were inoculated into the detoxification pretreatment solution and soaked for 24 hours at 25°C in the dark;

[0011] The detoxification pretreatment solution consists of MS basal medium, ribavirin 100-120 mg / L, triazole nucleoside 80-90 mg / L, and sucrose 20 g / L, and the pH of the detoxification pretreatment solution is adjusted to 5.8;

[0012] Preferably, the detoxification pretreatment solution is formulated as follows: MS basal medium + ribavirin 110 mg / L + triazole nucleoside 85 mg / L + sucrose 20 g / L, pH 5.8;

[0013] The preferred components (per liter) of the MS basal medium are: NH4NO3 1650 mg / L, KNO3 1900 mg / L, KH2PO4 170 mg / L, MgSO4·7H2O 370 mg / L, CaCl2·2H2O 440 mg / L, KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, FeSO4·7H2O 27.8 mg / L, Na2-EDTA 37.3 mg / L, inositol 100 mg / L, glycine 2 mg / L, and vitamin B1. 0.1 mg / L, Vitamin B6 0.5 mg / L, Niacin 0.5 mg / L.

[0014] S3 Differentiation Induction Culture

[0015] The detoxified apical buds from step S2 were inoculated into differentiation induction medium and cultured under set conditions for 12 days to obtain clustered buds. The differentiation induction medium consisted of WPM medium, 0.8-1.0 mg / L of 6-benzylaminopurine (6-BA), 0.6-0.8 mg / L of indolebutyric acid (IBA), 25 g / L of sucrose, and 6 g / L of agar, with the pH adjusted to 5.8-6.0. Preferably, the formulation of the differentiation induction medium was: WPM medium + 0.9 mg / L of 6-benzylaminopurine + 0.7 mg / L of indolebutyric acid + 25 g / L of sucrose + 6 g / L of agar, with a pH of 5.9. The preferred components (per liter) of the WPM medium were: 0.54 g / L of NH4NO3, 0.065 g / L of CaCl2·2H2O, and MgSO4·7H2O. 0.85 g / L, KH₂PO₄ 0.74 g / L, MnSO₄·4H₂O 25.6 mg / L, ZnSO₄·7H₂O 10.5 mg / L, H₃BO₃ 7.8 mg / L, NaMoO₄·2H₂O 0.58 mg / L, CuSO₄·5H₂O 0.049 mg / L, CoCl₂·6H₂O 0.072 mg / L, KI 0.07 mg / L, nicotinic acid 0.68 mg / L, vitamin B1 1.58 mg / L, glycine 5.23 mg / L, vitamin B6 0.87 mg / L, inositol 100 mg / L, FeSO₄·7H₂O 28.75 mg / L, Na2-EDTA 33.25 mg / L; the differentiation induction culture conditions were set as follows: culture temperature 25℃, culture humidity 60%, light intensity 2000 Lx, and light duration 14 h / d.

[0016] S4 propagation and subculture

[0017] The clustered shoots obtained in step S3 were divided into single shoots and inoculated into a proliferation subculture medium. Under set conditions, the shoots were cultured for 30 days to obtain proliferating shoots. The proliferation subculture medium consisted of 1 / 2 MS medium, 0.5 mg / L of 6-benzylaminopurine, 0.2 mg / L of naphthaleneacetic acid (NAA), 0.4 g / L of hydrolyzed casein, 25 g / L of sucrose, and 5 g / L of agar, with the pH adjusted to 5.8. Preferably, the pH of the proliferation subculture medium was adjusted using a 1 mol / L hydrochloric acid or sodium hydroxide solution. The preferred components (per liter) of the 1 / 2 MS medium were: 825 mg / L NH4NO3, 950 mg / L KNO3, 85 mg / L KH2PO4, 185 mg / L MgSO4·7H2O, 220 mg / L CaCl2·2H2O, 0.83 mg / L KI, 6.2 mg / L H3BO3, and MnSO4·4H2O. The following ingredients were added: 22.3 mg / L ZnSO4・7H2O 8.6 mg / L Na2MoO4・2H2O 0.25 mg / L CuSO4・5H2O 0.025 mg / L CoCl2・6H2O 0.025 mg / L FeSO4・7H2O 27.8 mg / L Na2-EDTA 37.3 mg / L inositol 100 mg / L glycine 2 mg / L, vitamin B1 0.1 mg / L, vitamin B6 0.5 mg / L, and niacin 0.5 mg / L. The conditions for the propagation and subculture were: culture temperature 23℃, culture humidity 45%, light intensity 2500 Lx, and photoperiod 16 h / d.

[0018] S5 rooting culture

[0019] Healthy, proliferating shoots with a height of 2-3 cm were selected and inoculated into a rooting medium. Under set conditions, rooted seedlings were obtained after 25 days of cultivation. The rooting medium consisted of 1 / 3 MS medium, indolebutyric acid (IBA) 0.1-0.12 mg / L, activated carbon 500 mg / L, sucrose 25 g / L, and agar 5 g / L, with the pH adjusted to 6.0. Preferably, the rooting medium formulation was: 1 / 3 MS medium + indolebutyric acid 0.1 mg / L + activated carbon 500 mg / L + sucrose 25 g / L + agar 5 g / L, pH 6.0. The preferred components of the 1 / 3 MS medium were those where the concentrations of macro- and micro-elements were reduced to 1 / 3 of the preferred components of the MS basic medium, while the organic components remained unchanged. The rooting culture conditions were: culture temperature 25℃, culture humidity 60%, light intensity 2500 Lx, and photoperiod 16 h / d.

[0020] S6 domestication and transplanting

[0021] 1. Hardening off seedlings: Move the culture bottles of rooted seedlings to a greenhouse and harden off the seedlings for 5 days under the conditions of 25℃ and natural diffused light. Then open the bottles and add sterile water to the culture bottles up to 0.5cm above the surface of the culture medium, and continue hardening off the seedlings for 3 days.

[0022] 2. Disinfection and Transplanting: Remove the rooted seedlings and carefully wash off the culture medium adhering to the roots (avoid damaging the root system). Soak them in a 0.8% potassium permanganate solution for 3 minutes. Transplant the disinfected rooted seedlings into a sterilized substrate, which is a mixture of vermiculite, garden soil, and humus in a volume ratio of 1:1:1. Preferably, the substrate is sterilized by high-pressure steam at 121°C and 0.1 MPa for 20 minutes. After transplanting, cover with plastic film to maintain a humidity of 85%, a culture temperature of 25°C, and a light intensity of 3000 Lx.

[0023] 3. Post-transplanting management: Remove the plastic film 7 days after transplanting, gradually increase the light to full light, and spray 1 / 2MS nutrient solution regularly, preferably once every 3 days. The amount of spray should be enough to wet the leaf surface without dripping. Continue cultivation for 15 days to complete the acclimatization and transplanting.

[0024] Beneficial effects

[0025] 1. This invention optimizes the formulation of the terminal bud virus-free pretreatment solution for the physiological characteristics of Huaniu apple rootstock, and combines the synergistic detoxification effect of ribavirin and triazole nucleoside to effectively remove viruses from the terminal buds of the rootstock, with a detoxification rate of over 95%, significantly improving the germplasm purity of the rootstock.

[0026] 2. The differentiation induction medium used WPM as the basal medium, which is suitable for the nutritional requirements of the terminal buds of the *Prunus armeniaca* rootstock. The ratio of 6-BA to IBA can promote the differentiation of terminal buds, with a differentiation survival rate of over 85% and a proliferation coefficient of 5.0.

[0027] 3. The rooting medium is based on 1 / 3 MS, with reduced salt ion concentration, and combined with appropriate concentrations of IBA and activated carbon to avoid excessive callus proliferation. The rooting rate reaches over 90%, and the roots are robust and uniform.

[0028] 4. The acclimatization and transplanting process involves phased hardening of seedlings and optimization of substrate ratio, resulting in a transplant survival rate of over 88%, thus enabling large-scale propagation of virus-free seedlings from Huaniu apple rootstock. Detailed Implementation

[0029] I. Preparation and Sterilization Pretreatment of Basal Culture Medium Stock Solution

[0030] (a) MS basal medium stock solution (10-fold concentration)

[0031] Mother liquor containing macroelements: NH4NO3 16.5 g / L, KNO3 19.0 g / L, KH2PO4 1.7 g / L, MgSO4·7H2O 3.7 g / L, CaCl2·2H2O 4.4 g / L;

[0032] Trace element stock solution: KI 8.3 mg / L, H3BO3 62 mg / L, MnSO4·4H2O 223 mg / L, ZnSO4·7H2O 86 mg / L, Na2MoO4·2H2O 2.5 mg / L, CuSO4·5H2O 0.25 mg / L, CoCl2·6H2O 0.25 mg / L;

[0033] Iron salt mother liquor: FeSO4・7H2O 278mg / L, Na2-EDTA 373mg / L (prepare fresh and store away from light);

[0034] Organic component mother liquor: inositol 10g / L, glycine 200mg / L, vitamin B1 (thiamine hydrochloride) 10mg / L, vitamin B6 (pyridoxine hydrochloride) 50mg / L, niacin 50mg / L.

[0035] (ii) WPM culture medium stock solution (10-fold concentration)

[0036] Mother liquor containing macroelements: NH4NO3 5.4 g / L, CaCl2·2H2O 0.65 g / L, MgSO4·7H2O 8.5 g / L, KH2PO4 7.4 g / L;

[0037] The mother liquor containing trace elements, iron salts, and organic components is the same as that in MS basal medium (concentration unchanged).

[0038] (iii) Sterilization of culture medium

[0039] After all culture media are prepared, dispense them into 100mL glass culture bottles (25mL of culture media per bottle), place them in a vertical autoclave, and sterilize them at 121℃ and 0.1MPa pressure for 20 minutes. After sterilization, remove them and place them in a sterile room to cool to room temperature for later use.

[0040] II. Implementation Examples

[0041] Example 1: Virus-free tissue culture group of terminal buds from *Prunus armeniaca* rootstock using conventional parameters

[0042] A method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock, the specific steps of which are as follows:

[0043] Explant selection and pretreatment were carried out on sunny mornings in April and May. The terminal buds of the current year's new shoots of healthy, vigorous apple rootstock plants free from visible pests and diseases were selected. The terminal bud segments were cut into 1.5cm lengths, the outer old leaves were removed, and the inner two young leaves were retained. The explants were first rinsed with running tap water for 30 minutes to remove surface dirt and impurities, and then rinsed three times with sterile water (1 minute each time). The treated terminal buds were placed in a clean bench for later use.

[0044] Disinfection and detoxification of apical buds (1) Disinfection: In a clean bench, place the pretreated apical buds into a sterile conical flask, add 75% ethanol solution and soak for 30 seconds, rinse 3 times with sterile water; after pouring out the sterile water, add 0.1% mercuric chloride solution (add 1 drop of Tween-80), stir with a magnetic stirrer for 6 minutes (120 r / min), rinse 5 times with sterile water, and finally dry the surface moisture of the apical buds with sterile filter paper; (2) Detoxification: Prepare a detoxification pretreatment solution (take 100 mL of 10 times concentrated MS basic culture medium stock solution, add 110 mg ribavirin, 85 mg triazole nucleoside, 20 g sucrose, and make up to 1 L with deionized water, and adjust the pH to 5.8 with 1 mol / L hydrochloric acid); inoculate the disinfected apical buds into the detoxification pretreatment solution and soak for 24 h at 25°C in complete darkness.

[0045] Differentiation induction culture was prepared by adding 100 mL of 10x concentrated WPM medium stock solution, 0.9 mg 6-BA, 0.7 mg IBA, 25 g sucrose, and 6 g agar to a final volume of 1 L with deionized water, and adjusting the pH to 5.9 with 1 mol / L hydrochloric acid. One virus-free apical bud was inoculated into each bottle and placed in an artificial climate incubator for cultivation under the following conditions: 25℃, 60% humidity, 2000 Lx light intensity, and 14 h / d photoperiod. After 12 days of cultivation, clustered buds were obtained, with an apical bud differentiation survival rate of 87%.

[0046] Proliferation and subculture were performed using a culture medium prepared by adding 50 mL of 10-fold concentrated MS basal medium stock solution, 0.5 mg 6-BA, 0.2 mg NAA, 0.4 g hydrolyzed casein, 25 g sucrose, and 5 g agar to a final volume of 1 L with deionized water, and adjusting the pH to 5.8 with 1 mol / L hydrochloric acid. The clustered shoots were divided into single shoots, with one shoot inoculated per bottle. The culture conditions were: 23℃, 45% humidity, 2500 Lx light intensity, and a photoperiod of 16 h / d. After 30 days of culture, proliferating shoots were obtained with a proliferation coefficient of 5.2.

[0047] Rooting culture medium was prepared by taking 33.3 mL of 10-fold concentrated MS basal medium stock solution, adding 0.1 mg IBA, 500 mg activated carbon, 25 g sucrose, and 5 g agar, and bringing the volume to 1 L with deionized water, and adjusting the pH to 6.0 with 1 mol / L NaOH. Healthy proliferating shoots of 2-3 cm were selected, and one shoot was inoculated into each bottle. The culture conditions were: 25℃, 60% humidity, light intensity of 2500 Lx, and photoperiod of 16 h / d. Rooted seedlings were obtained after 25 days of culture, with a rooting rate of 92%.

[0048] Acclimatization and Transplanting (1) Hardening off: The rooted seedling culture bottles were moved to the greenhouse and hardened off for 5 days under natural diffused light at 25℃. After opening the bottles, sterile water was added to 0.5cm above the surface of the culture medium and hardened off for another 3 days. (2) Disinfection before transplanting: The rooted seedlings were taken out, the culture medium on the roots was washed, and the roots were soaked in 0.8% potassium permanganate solution for 3 minutes and rinsed with sterile water once. (3) Sterilization of substrate and transplanting: Vermiculite: garden soil: humus = 1:1:1 mixed substrate was sterilized at 121℃ and 0.1MPa for 20 minutes and put into nutrient pots. One rooted seedling was transplanted into each pot and covered with plastic film to maintain humidity of 85%, temperature of 25℃ and light intensity of 3000Lx. (4) Post-transplanting management: After 7 days, the film was removed and the light was gradually increased to full light. 1 / 2MS nutrient solution was sprayed once every 3 days (to moisten the leaves without dripping). After 15 days of cultivation, the survival rate of transplanting was 89%.

[0049] Example 2: High-concentration virus-free agent parameter group for tissue culture of terminal buds of *Prunus armeniaca* rootstock.

[0050] The operation steps in this embodiment are the same as in Embodiment 1, except that the following parameters are adjusted:

[0051] Detoxification pretreatment solution: MS basal medium + ribavirin 120 mg / L + triazole nucleoside 90 mg / L + sucrose 20 g / L (pH 5.8).

[0052] Differentiation induction medium: WPM medium + 6-BA 1.0 mg / L + IBA 0.8 mg / L + sucrose 25 g / L + agar 6 g / L (pH 6.0);

[0053] Rooting medium: 1 / 3 MS medium + IBA 0.12 mg / L + activated carbon 500 mg / L + sucrose 25 g / L + agar 5 g / L (pH 6.0).

[0054] Statistical results: virus elimination rate 96%, apical bud differentiation survival rate 86%, proliferation coefficient 5.0, rooting rate 89%, transplant survival rate 88%.

[0055] Example 3: Optimal Parameters for Virus-Free Tissue Culture of Terminal Buds from *Prunus armeniaca* Rootstock

[0056] This embodiment optimizes the parameter combination, and differs from Embodiment 1 in the following ways:

[0057] Explant: Terminal bud length 2.0cm, retaining 3 inner young leaves;

[0058] Culture medium pH: Differentiation induction medium pH 5.8, proliferation subculture medium pH 5.8, rooting medium pH 6.0;

[0059] Transplanting: After the substrate is sterilized, cool it to 25°C before transplanting.

[0060] Statistical results: virus elimination rate 97%, apical bud differentiation survival rate 88%, proliferation coefficient 5.3, rooting rate 93%, transplant survival rate 90%.

[0061] III. Comparative Example

[0062] Compared with the control group of Example 1 that did not undergo detoxification treatment

[0063] Except for the lack of detoxification treatment, the other steps, formulas, and conditions were the same as in Example 1.

[0064] Statistical results: virus elimination rate 30%, apical bud differentiation survival rate 85%, proliferation coefficient 5.1, rooting rate 90%; disease and pest incidence rate 40% 30 days after transplanting, transplant survival rate 65%.

[0065] Comparative Example 2: The control group using MS medium as the basis for differentiation induction.

[0066] The differentiation induction medium was replaced with: MS basal medium + 6-BA 0.9 mg / L + IBA 0.7 mg / L + sucrose 25 g / L + agar 6 g / L (pH 5.9), and the rest was the same as in Example 1.

[0067] Statistical results: Detoxification rate 95%, terminal bud differentiation survival rate 62%, proliferation coefficient 2.8, rooting rate 88%, transplant survival rate 70% (MS high salt ions disrupt ion-hormone balance, increasing terminal bud browning rate).

[0068] Comparative Example 3: Control group using the existing M9 rootstock tissue culture method

[0069] The terminal buds of *Prunus armeniaca* rootstock were treated using the M9 rootstock tissue culture method described in application number CN201310046571. Core parameters:

[0070] Differentiation induction medium: MS + 6-BA 0.8 mg / L + NAA 0.5 mg / L + sucrose 25 g / L + agar 5 g / L;

[0071] Rooting medium: 1 / 3 MS + IBA 0.1 mg / L + sucrose 25 g / L + agar 5 g / L;

[0072] The cultivation conditions are as described in Example 1 of this patent.

[0073] Statistical results: virus elimination rate 0, apical bud differentiation survival rate 70%, proliferation coefficient 3.0, rooting rate 82%, transplant survival rate 80%.

[0074] IV. Data Statistics and Results Analysis

[0075] Table 1 Comparison of core indicators between each embodiment and the comparative example.

[0076] Group Detoxification rate (%) Terminal bud differentiation survival rate (%) Proliferation coefficient Rooting rate (%) Transplant survival rate (%) Example 1 95 87 5.2 92 89 Example 2 96 86 5.0 89 88 Example 3 97 88 5.3 93 90 Comparative Example 1 30 85 5.1 90 65 Comparative Example 2 95 62 2.8 88 70 Comparative Example 3 0 70 3.0 82 80

[0077] Table 2. Effects of different IBA concentrations on the proliferation and rooting of *Prunus hupehensis* rootstock.

[0078] IBA concentration (mg / L) Number of inoculated plants Number of rooted plants Rooting rate (%) Root system state description 0.08 50 40 80 The root system is thin and weak, with ≤3 roots and a root length ≤1.5cm. 0.10 (Example 1) 50 46 92 The root system is robust, with ≥4 roots, each 2-3 cm long, and without callus tissue. 0.12 (Example 2) 50 44 88 The root system is relatively thick, with 3-4 roots and a small amount of callus tissue. 0.15 50 42 84 Significant callus proliferation and root deformities were observed.

[0079] Table 3. Effects of different virus-removing agent ratios on the virus removal effect of terminal buds of *Prunus hupehensis* rootstock.

[0080] Detoxification pretreatment solution formulation (MS medium based) Number of plants treated Number of successfully detoxified strains Detoxification rate (%) Browning rate of apical buds (%) Ribavirin 100 mg / L + Triazole nucleoside 80 mg / L 50 46 92 5 Ribavirin 110 mg / L + Triazole nucleoside 85 mg / L (Example 1) 50 47 94 3 Ribavirin 120 mg / L + Triazole nucleoside 90 mg / L (Example 2) 50 48 96 4 Ribavirin 150 mg / L + Triazole nucleoside 100 mg / L 50 47 94 10

[0081] Results Analysis

[0082] Necessity of virus removal treatment: Comparative example 1 shows that although the lack of virus removal treatment does not affect the basic differentiation / proliferation / rooting indicators, the high incidence of diseases and pests after transplanting leads to a sharp drop in survival rate. Virus removal is the core to ensure the survival of tissue culture seedlings in the field.

[0083] Culture medium compatibility: Comparative example 2 verified that WPM culture medium is more suitable for the differentiation of terminal buds of *Prunus armeniaca* rootstock. The high salt ions in MS will disrupt the ion-hormone balance and inhibit differentiation and proliferation.

[0084] Hormone concentration optimization: IBA 0.1 mg / L is the optimal concentration for rooting (too high a concentration leads to callus proliferation, too low a concentration leads to weak roots); the detoxification formula of ribavirin 110 mg / L + triazole nucleoside 85 mg / L balances detoxification effect and apical bud survival rate.

[0085] Advantages of this invention: The indicators of Examples 1-3 are significantly better than the existing M9 rootstock tissue culture method (Comparative Example 3), proving that the optimization scheme of this invention for Huaniu apple rootstock has outstanding substantive features and significant progress.

[0086] V. Conclusion

[0087] The virus-free tissue culture system for Huaniu apple rootstock apical buds constructed in this invention achieves technical results of ≥95% virus-free rate, ≥86% apical bud differentiation survival rate, ≥5.0 proliferation coefficient, ≥89% rooting rate, and ≥88% transplant survival rate, significantly superior to existing apple rootstock tissue culture techniques. This method features controllable parameters and standardized operation, enabling large-scale propagation of virus-free Huaniu apple rootstock seedlings, providing high-quality rootstock support for the Huaniu apple industry, and possessing significant industrial applicability.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions of the present invention, or equivalent substitutions can be made to some technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the protection scope of the technical solutions of the present invention.

Claims

1. A method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock, characterized in that, Includes the following steps: S1 Explant Selection and Pretreatment: Select the terminal buds of healthy *Prunus armeniaca* rootstock plants, rinse with running water and then with sterile water, and set aside for use. S2 Apical Bud Disinfection and Detoxification Treatment: The pretreated apical buds were first disinfected with 75% ethanol (v / v) and 0.1% mercuric chloride (w / w) in sequence. Then, the disinfected apical buds were inoculated into the detoxification pretreatment solution and soaked in the dark for 24 hours. The detoxification pretreatment solution consisted of MS basal medium, ribavirin 100-120 mg / L, triazole nucleoside 80-90 mg / L, and sucrose 20 g / L, and the pH of the detoxification pretreatment solution was adjusted to 5.

8. S3 Differentiation Induction Culture: The apical buds detoxified in step S2 were inoculated into the differentiation induction medium and cultured under set conditions for 12 days to obtain clustered buds; the differentiation induction medium consisted of WPM medium, 0.8-1.0 mg / L of 6-benzylaminopurine, 0.6-0.8 mg / L of indolebutyric acid, 25 g / L of sucrose, and 6 g / L of agar, and the pH of the differentiation induction medium was adjusted to 5.8-6.0; S4 Proliferation and Subculture: The clustered shoots obtained in step S3 were divided into single shoots and inoculated into a proliferation and subculture medium. They were cultured under set conditions for 30 days to obtain proliferating shoots. The proliferation and subculture medium consisted of 1 / 2 MS medium, 0.5 mg / L of 6-benzylaminopurine, 0.2 mg / L of naphthaleneacetic acid, 0.4 g / L of hydrolyzed casein, 25 g / L of sucrose, and 5 g / L of agar. The pH of the proliferation and subculture medium was adjusted to 5.

8. S5 Rooting Culture: Selected proliferating shoots with a height of 2-3 cm were inoculated into rooting medium and cultured under set conditions for 25 days to obtain rooted seedlings; the rooting medium consisted of 1 / 3 MS medium, indolebutyric acid 0.1-0.12 mg / L, activated carbon 500 mg / L, sucrose 25 g / L, and agar 5 g / L, and the pH of the rooting medium was adjusted to 6.0; S6 Acclimatization and Transplanting: First, place the culture bottles of rooted seedlings in a greenhouse environment and close the bottles for 5 days to harden off the seedlings. Then, open the bottles and harden off the seedlings for 3 days. After that, take out the rooted seedlings and wash off the culture medium attached to their roots. After disinfecting with potassium permanganate solution, transplant them into sterilized substrate and keep them moist for 15 days. The substrate is made by mixing vermiculite, garden soil and humus in a volume ratio of 1:1:

1.

2. The method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock according to claim 1, characterized in that, The terminal bud mentioned in step S1 is the terminal bud at the tip of the current year's new shoot of the *Prunus armeniaca* rootstock. The terminal bud is 1.5-2.0 cm long and retains 2-3 young leaves in the inner layer of the terminal bud.

3. The method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock according to claim 1, characterized in that, The disinfection step in step S2 is as follows: the pretreated apical bud is placed in a 75% ethanol solution for 30 seconds and rinsed with sterile water 3 times; then the apical bud is placed in a 0.1% mercuric chloride solution, 1-2 drops of Tween-80 are added to the mercuric chloride solution, shaken for 6 minutes, rinsed with sterile water 5 times, and finally the surface moisture of the apical bud is dried with sterile filter paper.

4. The method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock according to claim 1, characterized in that, The conditions for differentiation induction culture in step S3 are: culture temperature 25℃, culture humidity 60%, light intensity 2000Lx, and light duration 14h / d.

5. The method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock according to claim 1, characterized in that, The conditions for proliferation and subculture in step S4 are: culture temperature 23℃, culture humidity 45%, light intensity 2500Lx, and light duration 16h / d.

6. The method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock according to claim 1, characterized in that, The pH of the proliferation and subculture medium described in step S4 is adjusted using a 1 mol / L hydrochloric acid or sodium hydroxide solution.

7. The method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock according to claim 1, characterized in that, The rooting culture conditions in step S5 are set as follows: culture temperature 25℃, culture humidity 60%, light intensity 2500Lx, and light duration 16h / d.

8. The method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock according to claim 1, characterized in that, The specific steps for acclimatization and transplanting in step S6 are as follows: (1) Hardening off seedlings: Move the culture bottles of the rooted seedlings to the greenhouse and harden off seedlings in the closed bottles for 5 days (temperature 25℃, natural diffused light); when hardening off seedlings in the open bottles, add sterile water to the culture bottles up to 0.5cm above the surface of the culture medium and continue hardening off seedlings for 3 days; (2) Disinfection and transplanting: Take out the rooted seedlings, wash the culture medium from the roots, and soak them in a 0.8% potassium permanganate solution for 3 minutes; transplant the disinfected rooted seedlings into the sterilized substrate, cover them with plastic film to maintain a humidity of 85%, a culture temperature of 25℃, and a light intensity of 3000Lx; (3) Post-transplanting management: Remove the plastic film 7 days after transplanting, gradually increase the light to full light, spray 1 / 2MS nutrient solution regularly, and continue to cultivate for 15 days.

9. The method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock according to claim 8, characterized in that, The sterilization method of the sterilization substrate in step S6 is high-pressure steam sterilization, and the sterilization conditions are 121℃ and 0.1MPa for 20 minutes.

10. The method for virus-free tissue culture using terminal buds of *Prunus armeniaca* rootstock according to claim 8, characterized in that, The 1 / 2MS nutrient solution mentioned in step S6 is sprayed once every 3 days, and the amount sprayed is enough to wet the leaf surface without dripping.