A method for rapid propagation of peach rootstock with anti-yellowing properties through tissue culture

Abstract

This invention relates to the field of plant tissue culture technology, and in particular to a method for rapid propagation of peach rootstock for preventing yellowing, comprising the following steps: S1, selecting the top shoots of the peach rootstock for preventing yellowing, cleaning and disinfecting the top shoots, and then inoculating them into a differentiation induction medium for culture to obtain clustered shoots; S2, inoculating the clustered shoots into a proliferation subculture medium for culture to obtain proliferating shoots, and then inoculating the proliferating shoots into a rooting medium to induce rooting. The rapid propagation method provided by this invention, specifically for propagating peach rootstock for preventing yellowing, can quickly obtain excellent seedlings, shortening the seedling time and reducing seedling costs.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a method for rapid propagation of peach rootstock with anti-yellowing properties through tissue culture. Background Technology

[0002] Peach rootstock is an important component of peach tree cultivation. It consists of the rootstock and rhizome of the peach tree, also known as the rootstock branch, and plays a crucial role in supporting the tree, absorbing nutrients, regulating water, and facilitating nutrient transfer. The quality and selection of peach rootstock affect peach tree growth, fruit yield, and quality. It also influences the peach tree's disease resistance, cold tolerance, and adaptability. Improper selection can not only reduce the quality and yield of the fruit but also result in poor-quality fruit.

[0003] As a superior peach rootstock in my country, the demand for anti-yellowing peach rootstock is substantial, and its propagation and promotion play a crucial role in the development of the entire industry. Therefore, research on anti-yellowing peach rootstock urgently requires a more efficient and rapid propagation method, and asexual reproduction is one of the important approaches to solving this problem. Currently, there are numerous studies on the asexual reproduction of anti-yellowing peach rootstock, but methods such as grafting and cutting still cannot achieve the rapid propagation of a large number of superior plants.

[0004] Therefore, if a high-efficiency and stable rapid propagation system for anti-yellowing peach rootstocks can be established using superior anti-yellowing peach rootstocks as materials, it will be of vital importance to the development and application of anti-yellowing peach rootstocks in the fields of genetic improvement and new variety breeding. Summary of the Invention

[0005] The purpose of this invention is to provide a method for rapid propagation of anti-yellowing peach rootstock through tissue culture, so as to achieve rapid and large-scale propagation of superior germplasm of anti-yellowing peach rootstock, thereby providing strong technical support for the genetic improvement and industrial development of anti-yellowing peach rootstock.

[0006] To achieve the above objectives, the present invention provides a method for rapid propagation of peach rootstock with anti-yellowing properties through tissue culture, comprising the following steps:

[0007] S1. Select the top shoots of the anti-yellowing peach rootstock, clean and disinfect the top shoots, and then inoculate them into the differentiation induction medium for culture to obtain clustered shoots;

[0008] S2. Inoculate the clustered shoots into the proliferation subculture medium, culture them to obtain proliferating shoots, and then inoculate the proliferating shoots into the rooting medium to induce rooting.

[0009] In this invention, the top bud in S1 is the top bud of a healthy, disease-free, and pest-free peach rootstock.

[0010] In this invention, the disinfection process in S1 includes: disinfecting the top buds with an ethanol solution, washing them, disinfecting them again with a mercuric chloride solution, and washing them a second time.

[0011] In this invention, the top buds after secondary washing are placed on the surface of sterile filter paper and the moisture is absorbed.

[0012] In this invention, the volume concentration of the ethanol solution is 70%-80%, the disinfection time of the ethanol solution is 20-40 seconds, the mass concentration of the mercuric chloride solution is 0.05%-0.15%, and the disinfection time of the mercuric chloride solution is 5-7 minutes.

[0013] In this invention, the differentiation induction medium in S1 includes MS medium + 6-benzylaminopurine 0.11-1 mg / L + indolebutyric acid 0.21-1 mg / L + sucrose 20-30 g / L and agar 6-7 g / L.

[0014] In this invention, the components of the differentiation-inducing culture medium are preferably:

[0015] NH4NO3 0.55 g / L, CaCl2·2H2O 0.057 g / L, MgSO4·7H2O 0.76 g / L, KH2PO4 0.62 g / L, MnSO4·4H2O 24.5 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 5.5 mg / L, NaMoO4·2H2O 0.54 mg / L, CuSO4·5H2O 0.043 mg / L, CoCl2·6H2O 0.026 mg / L, KI 0.05 mg / L, nicotinic acid 0.45 mg / L, vitamin B1 1.12 mg / L, glycine 3.15 mg / L, vitamin B6 0.54 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.25 mg / L, 6-benzylaminopurine 0.11-1 mg / L, indolebutyric acid 0.21-1 mg / L, sucrose 25 g / L, agar 6 g / L.

[0016] In this invention, the culture temperature in S1 is 18-25℃, the culture time is 7-14 days, and the culture pH is 5.8-6.

[0017] In this invention, the proliferation and subculture medium in S2 is 1 / 2 MS medium.

[0018] In this invention, the preferred components of the 1 / 2MS culture medium are:

[0019] NH4NO3 825mg / L, KNO3 950mg / L, KH2PO4 85mg / L, MgSO4·7H2O 185mg / L, CaCl2·2H2O 220mg / L, KI 0.83mg / L, H3BO3 6.2mg / L, MnSO4·4H2O 22.3mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, FeSO4·7H2O 27.8mg / L, Na2-EDTA 37.3mg / L, inositol 100mg / L, glycine 2mg / L, vitamin B1 0.1 mg / L, Vitamin B6 0.5 mg / L, Niacin 0.5 mg / L.

[0020] In this invention, the culture temperature in S2 is 18-25℃, the humidity is 40%-50%, and the culture period is 30-35 days.

[0021] In this invention, the rooting medium in S2 includes MS medium + 0.04-1 mg / L of 6-benzylaminopurine + 0.55-1 mg / L of indolebutyric acid + 20-30 g / L of sucrose + 6-7 g / L of agar.

[0022] In this invention, the components of the rooting culture medium in S2 are preferably:

[0023] NH4NO3 0.25 g / L, CaCl2·2H2O 0.076 g / L, MgSO4·7H2O 0.52 g / L, KH2PO4 0.46 g / L, MnSO4·4H2O 19.5 mg / L, ZnSO4·7H2O 9.8 mg / L, H3BO3 5.87 mg / L, NaMoO4·2H2O 0.43 mg / L, CuSO4·5H2O 0.034 mg / L, CoCl2·6H2O 0.034 mg / L, KI 0.04 mg / L, nicotinic acid 0.46 mg / L, vitamin B1 1.35 mg / L, glycine 3.25 mg / L, vitamin B6 0.6 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.65 mg / L, 6-benzylaminopurine 0.04-1 mg / L, indolebutyric acid 0.55-1 mg / L, sucrose 25 g / L, agar 6 g / L.

[0024] In this invention, the temperature for inducing rooting in S2 is 23-27℃, the humidity for inducing rooting is 50%-70%, and the number of days for inducing rooting is 20-35 days.

[0025] In this invention, the differentiation induction medium, proliferation subculture medium, and rooting medium mentioned above all need to be sterilized before use. The sterilization process includes: placing the differentiation induction medium, proliferation subculture medium, and rooting medium in an autoclave and sterilizing them at 120-122°C for 20-40 minutes.

[0026] The present invention has the following beneficial effects:

[0027] This invention provides a method for rapid propagation of anti-yellowing peach rootstock through tissue culture, comprising the following steps: S1, selecting the top shoots of the anti-yellowing peach rootstock, cleaning and disinfecting the top shoots, and then inoculating them into a differentiation induction medium for culture to obtain clustered shoots; S2, inoculating the clustered shoots into a proliferation subculture medium for culture to obtain proliferating shoots, and then inoculating the proliferating shoots into a rooting medium to induce rooting. The rapid propagation method provided by this invention, specifically for the propagation of anti-yellowing peach rootstock, can quickly obtain excellent seedlings, shortening the seedling time and reducing seedling costs. Experiments have shown that the rapid propagation method for anti-yellowing peach rootstock through tissue culture provided by this invention achieves a top shoot differentiation induction survival rate of over 80%, a shoot proliferation coefficient of up to 5, and a rooting rate of over 90%, realizing the rapid propagation of anti-yellowing peach rootstock.

[0028] This invention further specifies that the differentiation-inducing medium comprises MS medium + 0.11-1 mg / L 6-benzylaminopurine + 0.21-1 mg / L indolebutyric acid + 20-30 g / L sucrose and 6-7 g / L agar. MS medium provides the core nutrients such as nitrogen, phosphorus, potassium, calcium, and magnesium for the growth of the apical buds, meeting the basic material requirements for cell division and growth, and laying the foundation for the bud differentiation nutritional environment. 6-Benzylaminopurine promotes the division and proliferation of bud cells, induces active meristems, stimulates the germination of lateral buds, and increases the number of clustered buds, thereby improving subsequent proliferation efficiency. Indolebutyric acid, as an auxin regulator, synergizes with 6-benzylaminopurine to promote cell elongation, balance the excessive cell division induced by 6-benzylaminopurine, avoid imbalance in bud growth, and provide directional guidance for subsequent bud morphology formation.

[0029] This invention further specifies that the rooting medium comprises MS medium + 0.04-1 mg / L 6-benzylaminopurine + 0.55-1 mg / L indolebutyric acid + 20-30 g / L sucrose + 6-7 g / L agar. Indolebutyric acid, as an auxin regulator, can promote vascular tissue differentiation, induce root primordia formation, and stimulate cell elongation, accelerating the elongation and growth of adventitious roots. 0.04-1 mg / L of 6-benzylaminopurine can activate meristematic cell activity, promote root primordia differentiation, and simultaneously prevent excessive callus proliferation, ensuring a stable and efficient rooting process.

[0030] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0031] Figure 1 This is a diagram of the growth status of clustered buds in Example 1. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. The features mentioned above or in the specific examples mentioned in this invention can be combined arbitrarily, and these specific embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention.

[0033] Example 1

[0034] Prepare a differentiation induction medium, the components of which include:

[0035] NH4NO3 0.55 g / L, CaCl2·2H2O 0.057 g / L, MgSO4·7H2O 0.76 g / L, KH2PO4 0.62 g / L, MnSO4·4H2O 24.5 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 5.5 mg / L, NaMoO4·2H2O 0.54 mg / L, CuSO4·5H2O 0.043 mg / L, CoCl2·6H2O 0.026 mg / L, KI 0.05 mg / L, nicotinic acid 0.45 mg / L, vitamin B1 1.12 mg / L, glycine 3.15 mg / L, vitamin B6 0.54 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.25 mg / L, 6-benzylaminopurine 1 mg / L, indolebutyric acid 1 mg / L, sucrose 25 g / L, agar 6 g / L.

[0036] Prepare a proliferation and subculture medium. The components of the proliferation and subculture medium include:

[0037] NH4NO3 825mg / L, KNO3 950mg / L, KH2PO4 85mg / L, MgSO4·7H2O 185mg / L, CaCl2·2H2O 220mg / L, KI 0.83mg / L, H3BO3 6.2mg / L, MnSO4·4H2O 22.3mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, FeSO4·7H2O 27.8mg / L, Na2-EDTA 37.3mg / L, inositol 100mg / L, glycine 2mg / L, vitamin B1 0.1mg / L, vitamin B6 0.5 mg / L, nicotinic acid 0.5 mg / L.

[0038] Prepare a rooting medium, the components of which include:

[0039] NH4NO3 0.25 g / L, CaCl2·2H2O 0.076 g / L, MgSO4·7H2O 0.52 g / L, KH2PO4 0.46 g / L, MnSO4·4H2O 19.5 mg / L, ZnSO4·7H2O 9.8 mg / L, H3BO3 5.87 mg / L, NaMoO4·2H2O 0.43 mg / L, CuSO4·5H2O 0.034 mg / L, CoCl2·6H2O 0.034 mg / L, KI 0.04 mg / L, nicotinic acid 0.46 mg / L, vitamin B1 1.35 mg / L, glycine 3.25 mg / L, vitamin B6 0.6 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.65 mg / L, 6-benzylaminopurine 0.5 mg / L, indolebutyric acid 0.55 mg / L, sucrose 25 g / L, agar 6 g / L.

[0040] After preparation, place the above differentiation induction medium, proliferation subculture medium and rooting medium in an autoclave and sterilize at 121°C for 20 minutes.

[0041] A method for rapid propagation of peach rootstock with anti-yellowing properties via tissue culture includes the following steps:

[0042] S1. After cleaning the top buds of healthy, disease-free, and pest-free peach rootstocks, place them in a clean bench and disinfect them with 75% ethanol solution for 30 seconds, wash them three times with sterile water, then disinfect them with 0.1% mercuric chloride solution for 6 minutes, wash them three times with sterile water, and then place them on sterile filter paper to absorb the surface moisture. The cleaned top buds are then inoculated into the prepared differentiation induction medium and cultured at 25℃ and pH 6 for 14 days to obtain clustered buds with a differentiation induction survival rate of 85%.

[0043] S2. The clustered shoots were inoculated into the proliferation subculture medium and cultured at 25℃ and 50% humidity for 35 days to obtain proliferating shoots with a shoot proliferation coefficient of 5. The proliferating shoots were then inoculated into the rooting medium and induced to root for 35 days at 25℃ and 50% humidity, with a rooting rate of 95%.

[0044] The clustered buds obtained in this embodiment are as follows: Figure 1 As shown, from Figure 1 It can be seen that multiple healthy new shoots differentiate around the tender shoot, and the number of shoots is obviously increased. Moreover, the leaves of the top tender shoot are unfolded and healthy in color after culture, indicating that the differentiation induction medium can effectively maintain the activity of the top tender shoot.

[0045] Example 2

[0046] Prepare a differentiation induction medium, the components of which include:

[0047] NH4NO3 0.55 g / L, CaCl2·2H2O 0.057 g / L, MgSO4·7H2O 0.76 g / L, KH2PO4 0.62 g / L, MnSO4·4H2O 24.5 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 5.5 mg / L, NaMoO4·2H2O 0.54 mg / L, CuSO4·5H2O 0.043 mg / L, CoCl2·6H2O 0.026 mg / L, KI 0.05 mg / L, nicotinic acid 0.45 mg / L, vitamin B1 1.12 mg / L, glycine 3.15 mg / L, vitamin B6 0.54 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.25 mg / L, 6-benzylaminopurine 0.8 mg / L, indolebutyric acid 0.6 mg / L, sucrose 25 g / L, agar 6 g / L.

[0048] Prepare a proliferation and subculture medium. The components of the proliferation and subculture medium include:

[0049] NH4NO3 825mg / L, KNO3 950mg / L, KH2PO4 85mg / L, MgSO4·7H2O 185mg / L, CaCl2·2H2O 220mg / L, KI 0.83mg / L, H3BO3 6.2mg / L, MnSO4·4H2O 22.3mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, FeSO4·7H2O 27.8mg / L, Na2-EDTA 37.3mg / L, inositol 100mg / L, glycine 2mg / L, vitamin B1 0.1mg / L, vitamin B6 0.5 mg / L, nicotinic acid 0.5 mg / L.

[0050] Prepare a rooting medium, the components of which include:

[0051] NH4NO3 0.25 g / L, CaCl2·2H2O 0.076 g / L, MgSO4·7H2O 0.52 g / L, KH2PO4 0.46 g / L, MnSO4·4H2O 19.5 mg / L, ZnSO4·7H2O 9.8 mg / L, H3BO3 5.87 mg / L, NaMoO4·2H2O 0.43 mg / L, CuSO4·5H2O 0.034 mg / L, CoCl2·6H2O 0.034 mg / L, KI 0.04 mg / L, nicotinic acid 0.46 mg / L, vitamin B1 1.35 mg / L, glycine 3.25 mg / L, vitamin B6 0.6 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.65 mg / L, 6-benzylaminopurine 0.2 mg / L, indolebutyric acid 0.6 mg / L, sucrose 25 g / L, agar 6 g / L.

[0052] After preparation, place the above differentiation induction medium, proliferation subculture medium and rooting medium in an autoclave and sterilize at 121°C for 20 minutes.

[0053] A method for rapid propagation of peach rootstock with anti-yellowing properties via tissue culture includes the following steps:

[0054] S1. After cleaning the top buds of healthy, disease-free, and pest-free peach rootstocks, place them in a clean bench and disinfect them with 75% ethanol solution for 30 seconds, wash them three times with sterile water, then disinfect them with 0.1% mercuric chloride solution for 6 minutes, wash them three times with sterile water, and then place them on sterile filter paper to absorb the surface moisture. The cleaned top buds are then inoculated into the prepared differentiation induction medium and cultured at 25℃ and pH 6 for 14 days to obtain clustered buds with a differentiation induction survival rate of 80%.

[0055] S2. The clustered shoots were inoculated into the proliferation subculture medium and cultured at 25℃ and 50% humidity for 35 days to obtain proliferating shoots with a shoot proliferation coefficient of 5. The proliferating shoots were then inoculated into the rooting medium and induced to root for 35 days at 25℃ and 50% humidity, with a rooting rate of 93%.

[0056] Comparative Example 1

[0057] Prepare a differentiation induction medium, the components of which include:

[0058] NH4NO3 0.55 g / L, CaCl2·2H2O 0.057 g / L, MgSO4·7H2O 0.76 g / L, KH2PO4 0.62 g / L, MnSO4·4H2O 24.5 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 5.5 mg / L, NaMoO4·2H2O 0.54 mg / L, CuSO4·5H2O 0.043 mg / L, CoCl2·6H2O 0.026 mg / L, KI 0.05 mg / L, nicotinic acid 0.45 mg / L, vitamin B1 1.12 mg / L, glycine 3.15 mg / L, vitamin B6 0.54 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.25 mg / L, indolebutyric acid 1 mg / L, sucrose 25 g / L, agar 6 g / L.

[0059] Prepare a proliferation and subculture medium. The components of the proliferation and subculture medium include:

[0060] NH4NO3 825mg / L, KNO3 950mg / L, KH2PO4 85mg / L, MgSO4·7H2O 185mg / L, CaCl2·2H2O 220mg / L, KI 0.83mg / L, H3BO3 6.2mg / L, MnSO4·4H2O 22.3mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, FeSO4·7H2O 27.8mg / L, Na2-EDTA 37.3mg / L, inositol 100mg / L, glycine 2mg / L, vitamin B1 0.1mg / L, vitamin B6 0.5 mg / L, nicotinic acid 0.5 mg / L.

[0061] Prepare a rooting medium, the components of which include:

[0062] NH4NO3 0.25 g / L, CaCl2·2H2O 0.076 g / L, MgSO4·7H2O 0.52 g / L, KH2PO4 0.46 g / L, MnSO4·4H2O 19.5 mg / L, ZnSO4·7H2O 9.8 mg / L, H3BO3 5.87 mg / L, NaMoO4·2H2O 0.43 mg / L, CuSO4·5H2O 0.034 mg / L, CoCl2·6H2O 0.034 mg / L, KI 0.04 mg / L, nicotinic acid 0.46 mg / L, vitamin B1 1.35 mg / L, glycine 3.25 mg / L, vitamin B6 0.6 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.65 mg / L, indolebutyric acid 0.55 mg / L, sucrose 25 g / L, agar 6 g / L.

[0063] After preparation, place the above differentiation induction medium, proliferation subculture medium and rooting medium in an autoclave and sterilize at 121°C for 20 minutes.

[0064] A method for rapid propagation of peach rootstock with anti-yellowing properties via tissue culture includes the following steps:

[0065] S1. After cleaning the top buds of healthy, disease-free, and pest-free peach rootstocks, place them in a clean bench. First, disinfect them with a 75% ethanol solution for 30 seconds, then wash them three times with sterile water. Next, disinfect them with a 0.1% mercuric chloride solution for 6 minutes, and wash them three times with sterile water. Then, place them on sterile filter paper to absorb the surface moisture of the buds. Subsequently, inoculate the cleaned top buds into the prepared differentiation induction medium and culture them at 25℃ and pH 6 for 14 days to obtain clustered buds. The differentiation induction survival rate is 45%.

[0066] S2. The clustered shoots were inoculated into the proliferation subculture medium and cultured at 25℃ and 50% humidity for 35 days to obtain proliferating shoots with a shoot proliferation coefficient of 2. The proliferating shoots were then inoculated into the rooting medium and induced to root for 35 days at 25℃ and 50% humidity, with a rooting rate of 60%.

[0067] Comparative Example 2

[0068] Prepare a differentiation induction medium, the components of which include:

[0069] NH4NO3 0.55 g / L, CaCl2·2H2O 0.057 g / L, MgSO4·7H2O 0.76 g / L, KH2PO4 0.62 g / L, MnSO4·4H2O 24.5 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 5.5 mg / L, NaMoO4·2H2O 0.54 mg / L, CuSO4·5H2O 0.043 mg / L, CoCl2·6H2O 0.026 mg / L, KI 0.05 mg / L, nicotinic acid 0.45 mg / L, vitamin B1 1.12 mg / L, glycine 3.15 mg / L, vitamin B6 0.54 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.25 mg / L, 6-benzylaminopurine 1 mg / L, sucrose 25 g / L, agar 6 g / L.

[0070] Prepare a proliferation and subculture medium. The components of the proliferation and subculture medium include:

[0071] NH4NO3 825mg / L, KNO3 950mg / L, KH2PO4 85mg / L, MgSO4·7H2O 185mg / L, CaCl2·2H2O 220mg / L, KI 0.83mg / L, H3BO3 6.2mg / L, MnSO4·4H2O 22.3mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, FeSO4·7H2O 27.8mg / L, Na2-EDTA 37.3mg / L, inositol 100mg / L, glycine 2mg / L, vitamin B1 0.1mg / L, vitamin B6 0.5 mg / L, nicotinic acid 0.5 mg / L.

[0072] Prepare a rooting medium, the components of which include:

[0073] NH4NO3 0.25 g / L, CaCl2·2H2O 0.076 g / L, MgSO4·7H2O 0.52 g / L, KH2PO4 0.46 g / L, MnSO4·4H2O 19.5 mg / L, ZnSO4·7H2O 9.8 mg / L, H3BO3 5.87 mg / L, NaMoO4·2H2O 0.43 mg / L, CuSO4·5H2O 0.034 mg / L, CoCl2·6H2O 0.034 mg / L, KI 0.04 mg / L, nicotinic acid 0.46 mg / L, vitamin B1 1.35 mg / L, glycine 3.25 mg / L, vitamin B6 0.6 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.65 mg / L, 6-benzylaminopurine 0.5 mg / L, sucrose 25 g / L, agar 6 g / L.

[0074] After preparation, place the above differentiation induction medium, proliferation subculture medium and rooting medium in an autoclave and sterilize at 121°C for 20 minutes.

[0075] A method for rapid propagation of peach rootstock with anti-yellowing properties via tissue culture includes the following steps:

[0076] S1. After cleaning the top buds of healthy, disease-free, and pest-free peach rootstocks, place them in a clean bench and disinfect them with 75% ethanol solution for 30 seconds, wash them three times with sterile water, then disinfect them with 0.1% mercuric chloride solution for 6 minutes, wash them three times with sterile water, and then place them on sterile filter paper to absorb the surface moisture. The cleaned top buds are then inoculated into the prepared differentiation induction medium and cultured at 25℃ and pH 6 for 14 days to obtain clustered buds with a differentiation induction survival rate of 50%.

[0077] S2. The clustered shoots were inoculated into the proliferation subculture medium and cultured at 25℃ and 50% humidity for 35 days to obtain proliferating shoots with a shoot proliferation coefficient of 2.5. The proliferating shoots were then inoculated into the rooting medium and induced to root at 25℃ and 50% humidity for 35 days, with a rooting rate of 55%.

[0078] Compared to the differentiation induction and rooting media provided in Example 1, Comparative Example 1 lacked the addition of 6-benzylaminopurine, resulting in a decrease in differentiation induction survival rate, proliferation coefficient, and rooting rate. This is because the absence of 6-benzylaminopurine during the differentiation induction stage fails to effectively stimulate the activity of meristematic tissue in the apical buds, inhibiting cell division and reducing the number of clustered buds. Consequently, the differentiation induction survival rate is significantly lower than in Example 1, further leading to a decrease in the proliferation coefficient. During rooting induction, the lack of 6-benzylaminopurine prevents the activation of root primordia differentiation activity and makes it difficult to balance cell proliferation and differentiation during rooting, resulting in slow adventitious root formation and a rooting rate of only 60%.

[0079] Comparative Example 2 did not include indolebutyric acid (IBA) in the differentiation induction and rooting media provided in Example 1. The absence of IBA during the differentiation stage prevented cell elongation and disrupted the balance of excessive 6-benzylaminopurine proliferation, leading to an imbalance in shoot growth and a decrease in differentiation survival rate and proliferation efficiency. During the rooting induction stage, IBA is a key substance for inducing root primordium formation; its absence prevented normal root primordium formation, hindered adventitious root growth, and resulted in a rooting rate of only 55%.

[0080] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for rapid propagation of peach rootstock with anti-yellowing properties via tissue culture, characterized in that, Includes the following steps: S1. Select the top shoots of the anti-yellowing peach rootstock, clean and disinfect the top shoots, and then inoculate them into the differentiation induction medium for culture to obtain clustered shoots; S2. Inoculate the clustered shoots into the proliferation subculture medium, culture them to obtain proliferating shoots, and then inoculate the proliferating shoots into the rooting medium to induce rooting; The components of the differentiation-inducing medium in S1 were: NH4NO3 0.55 g / L, CaCl2·2H2O 0.057 g / L, MgSO4·7H2O 0.76 g / L, KH2PO4 0.62 g / L, MnSO4·4H2O 24.5 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 5.5 mg / L, Na2MoO4·2H2O 0.54 mg / L, CuSO4·5H2O 0.043 mg / L, CoCl2·6H2O 0.026 mg / L, KI 0.05 mg / L, nicotinic acid 0.45 mg / L, vitamin B1 1.12 mg / L, glycine 3.15 mg / L, vitamin B6 0.54 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.25 mg / L, 6-benzylaminopurine 0.11-1 mg / L, indolebutyric acid 0.21-1 mg / L, sucrose 25 g / L, agar 6 g / L; The components of the proliferation and subculture medium in S2 were: NH4NO3 825 mg / L, KNO3 950 mg / L, KH2PO4 85 mg / L, MgSO4·7H2O 185 mg / L, CaCl2·2H2O 220 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; The components of the rooting medium in S2 are: NH4NO3 0.25 g / L, CaCl2·2H2O 0.076 g / L, MgSO4·7H2O 0.52 g / L, KH2PO4 0.46 g / L, MnSO4·4H2O 19.5 mg / L, ZnSO4·7H2O 9.8 mg / L, H3BO3 5.87 mg / L, Na2MoO4·2H2O 0.43 mg / L, CuSO4·5H2O 0.034 mg / L, CoCl2·6H2O 0.034 mg / L, KI 0.04 mg / L, nicotinic acid 0.46 mg / L, vitamin B1 1.35 mg / L, glycine 3.25 mg / L, vitamin B6 0.6 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.65 mg / L, 6-benzylaminopurine 0.04-1 mg / L, indolebutyric acid 0.55-1 mg / L, sucrose 25 g / L, agar 6 g / L.

2. The method for rapid propagation of peach rootstock with anti-yellowing properties according to claim 1, characterized in that, The top buds in S1 are the top buds of healthy, disease-free, and pest-free peach rootstocks that are resistant to yellowing.

3. The method for rapid propagation of peach rootstock with anti-yellowing properties according to claim 1, characterized in that, The disinfection process in S1 includes: disinfecting the top buds with ethanol solution, washing them, disinfecting them with mercuric chloride solution, and washing them a second time.

4. The method for rapid propagation of peach rootstock with anti-yellowing properties according to claim 3, characterized in that, The volume concentration of the ethanol solution is 70%-80%, and the disinfection time is 20-40 seconds. The mass concentration of the mercuric chloride solution is 0.05%-0.15%, and the disinfection time is 5-7 minutes.

5. The method for rapid propagation of peach rootstock with anti-yellowing properties according to claim 1, characterized in that, The culture temperature in S1 is 18-25℃, the culture time is 7-14 days, and the culture pH is 5.8-6.

6. The method for rapid propagation of peach rootstock with anti-yellowing properties according to claim 1, characterized in that, The culture temperature in S2 is 18-25℃, the humidity is 40%-50%, and the culture period is 30-35 days.

7. The method for rapid propagation of peach rootstock with anti-yellowing properties according to claim 1, characterized in that, The temperature for inducing rooting in S2 is 23-27℃, the humidity for inducing rooting is 50%-70%, and the number of days for inducing rooting is 20-35 days.

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