A method for tissue culture rapid propagation of a poor soil tolerant rootstock of apple

Abstract

The present application relates to the technical field of plant tissue culture, in particular to a method for tissue culture and rapid propagation of apple poor-tolerance rootstock, comprising the following steps: selecting mother trees continuously planted on poor mountain land for scion collection; washing the obtained stem segments and then soaking them in a bactericide, washing them clean, and then performing low-temperature juvenile treatment, cutting and sterilizing, and cutting off the browning wounds at both ends; placing the surface-sterilized explants into an anti-browning bud germination medium, culturing the clustered buds in a subculture proliferation medium, selecting sterile single seedlings and inoculating them into a low-salt rooting medium, performing dark culture and then light culture, and planting the rooted seedlings in a mixed substrate of grass charcoal and perlite, and then continuing to culture the seedlings after transplanting them into a simulated poor thin soil substrate to obtain rootstock seedlings. The present application selects the mother trees for scion collection on the poor mountain land in the north by using the above steps, effectively improves the activity and stress resistance of the explants by low-temperature juvenile treatment, and uses two kinds of substrates to gradually adapt the seedlings to the poor mountain soil environment, thereby greatly improving the poor-tolerance and stress resistance.

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 apple rootstocks tolerant of poor soil through tissue culture. Background Technology

[0002] The northern mountainous region is my country's core apple-producing area. It boasts ecological advantages such as abundant sunshine, large diurnal temperature variations, and excellent ventilation, resulting in apples with vibrant color, high sugar content, and superior quality, indicating significant potential for industry development. However, orchards in this region generally suffer from poor site conditions and severe soil infertility. Most mountainous areas have shallow soil layers, high sand and gravel content, scarce organic matter, and extremely poor water and fertilizer retention capacity. Furthermore, long-term soil erosion and wind erosion have led to continuous degradation of soil fertility, creating a typical barren and thin-soil planting environment that severely restricts the growth and development of apple trees.

[0003] Currently, apple cultivation in northern mountainous areas primarily utilizes conventional seedling rootstocks or ordinary grafted rootstocks. These rootstocks generally suffer from weak resistance to poor soil conditions and poor environmental adaptability. Under barren mountainous cultivation conditions, conventional rootstocks have insufficient root absorption capacity, resulting in weak tree growth, poor stress resistance, and problems such as slow growth, low fruit yield, and inconsistent quality. Existing apple rootstock propagation methods mainly rely on seed sowing, conventional cuttings, and ordinary tissue culture. Seed sowing has a long cycle and severe separation of seedling traits, making it impossible to stably retain the excellent traits of the mother tree that resist poor soil conditions. Cutting propagation has a low rooting rate and is difficult to mass-produce. Conventional tissue culture techniques generally suffer from severe browning of explants, and the seedlings produced are mostly suitable for fertile flatland planting environments. After transplanting to barren mountainous areas, they lack adaptability and their survival rate drops sharply, making it difficult to meet the production needs of the northern barren mountainous apple industry for a large supply of highly resistant, stable, high-quality, and robust seedlings. Based on this, a rapid propagation method for apple rootstocks adapted to the barren environment of northern mountainous areas was developed. Summary of the Invention

[0004] The purpose of this invention is to provide a method for rapid propagation of apple rootstocks tolerant to poor soil conditions through tissue culture. The method involves selecting robust, virus-free mother trees that have been continuously planted in poor mountainous areas of northern China and have stable traits of resistance to poor soil conditions for scion collection. Through low-temperature juvenile treatment, the activity and stress resistance of the explants are effectively enhanced. Two different substrates are used to allow the seedlings to gradually adapt to the poor, well-aerated, and nutrient-deficient mountainous soil environment, thereby significantly improving the seedlings' resistance to poor soil conditions and stress resistance.

[0005] To achieve the above objectives, this invention provides a method for rapid propagation of apple rootstocks tolerant to poor soil through tissue culture, comprising the following steps: S1. Select mother trees that have been continuously planted on barren mountain land, select mother trees that are vigorous, virus-free and resistant to barrenness for scion collection, and cut stem segments with 2-5 plump axillary buds. S2. After cleaning the stem segments obtained in S1, soak them in a fungicide for 10-20 minutes, rinse them clean, and then perform low-temperature aging treatment. After cutting the stem segments that have undergone low-temperature aging treatment, soak them in ethanol, rinse them with sterile water, and then sterilize them in mercuric chloride solution. After rinsing with sterile water, use sterile paper to absorb the moisture, and cut off the browned wounds at both ends to obtain surface-sterilized explants. S3. Prepare an anti-browning bud germination medium. Place the surface-sterilized explants from S2 into the anti-browning bud germination medium and culture them at 24-25℃ for 20-30 days with a light duration of 15-16 h / d. S4. Prepare a subculture proliferation medium. Under aseptic conditions, divide the clustered shoots of the explants cultured in S3. Retain 2-3 effective buds in each clustered shoot. Inoculate the clustered shoots into the subculture proliferation medium and culture for 20-30 days to obtain sterile single seedlings. S5. Prepare a low-salt rooting medium, select sterile single seedlings obtained in S4, remove the aged callus at the base, inoculate them into the low-salt rooting medium, first culture in the dark for 3-7 days and then culture in the light for 15-20 days to obtain rooted seedlings. S6. Leave the culture bottle cap in S5 half open for 2-5 days. Plant the rooted seedlings in a mixed substrate of peat moss and perlite for 12-18 days. After transplanting to a simulated barren soil substrate, continue to cultivate for 25-35 days to obtain rootstock seedlings with a plant height of 8-12cm.

[0006] Preferably, in S1, the mother tree is continuously planted on barren mountain land for 2-3 years, the scion harvesting time is in mid-April, and the stem segment length is 2-5cm.

[0007] Preferably, in S2, the cleaning process involves: rinsing with running water for 60-90 minutes, using a soft brush to remove impurities and fuzz from the surface of the stem segment, soaking in a detergent containing a neutral surfactant, and then rinsing with running water.

[0008] Preferably, in S2, the low-temperature aging treatment is as follows: the rinsed stem segments are wrapped in damp gauze and stored at 4°C in the dark for 6-8 days, and the length of the cut segments is 1-2 cm.

[0009] Preferably, in S2, the ethanol is 75% ethanol, the concentration of the mercuric chloride solution is 0.05-0.15%, and sterilization is performed in the mercuric chloride solution for 5-10 minutes.

[0010] Preferably, in S3, the anti-browning bud germination medium is: MS, 0.8 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L ascorbic acid, 200 mg / L hydrolyzed casein, 0.5 g / L PVP, 28 g / L sucrose and 6.5 g / L agar, with a pH of 5.6-5.8.

[0011] Preferably, in S4, the subculture medium is: MS, 1.0 mg / L 6-BA, 0.1 mg / L IBA, 80 mg / L phloroglucinol, 20 mg / L adenine, 30 mg / L sodium thiosulfate, 25 g / L sucrose and 6 g / L agar, with a pH of 5.6-5.8.

[0012] Preferably, in S5, the low-salt rooting medium consists of: 1 / 4 MS, 1.0 mg / L IBA, 0.1 mg / L NAA, 0.3 g / L activated carbon, 60 mg / L betaine, 50 mg / L humic acid, 18 g / L sucrose, and 5.5 g / L agar, with a pH of 5.5-5.8.

[0013] Preferably, in S6, the mixing ratio of peat moss and perlite is 2-5:1, and slow-release organic fertilizer accounting for 0.1-1% of the mass of the mixed substrate is added to the mixed substrate.

[0014] Preferably, in S6, the mass ratio of garden soil, river sand and weathered sandstone fragments in the barren thin soil matrix is ​​5-8:2-4:1, and humic acid aqueous solution is sprayed weekly during the cultivation process.

[0015] Therefore, the present invention employs the above-mentioned method for rapid propagation of apple rootstocks tolerant to poor soil through tissue culture, and its beneficial effects are as follows: 1. The tissue culture rapid propagation method provided by the present invention selects healthy and virus-free mother trees that have been continuously planted in barren mountainous areas of northern China and have stable barrenness resistance traits for scion collection. This method locks in the excellent barrenness resistance genetic traits of the rootstock from the source, and completely solves the problems of trait segregation and resistance degradation in traditional seedling cultivation. 2. The tissue culture rapid propagation method provided by this invention effectively enhances the activity and stress resistance of explants through low-temperature juvenile treatment. The cultivated rootstock seedlings are highly adaptable to the harsh environment of thin soil layer, nutrient deficiency and poor soil structure in northern mountainous areas. After planting, the root system develops well and the nutrient absorption capacity is strong, which can significantly improve the growth of apple trees in barren mountainous areas and improve the planting problems of weak trees, low yield and poor quality. 3. The tissue culture rapid propagation method provided by this invention uses two substrates to allow seedlings to gradually adapt to the barren, well-aerated, and nutrient-deficient mountain soil environment, which greatly improves the seedlings' resistance to barrenness and stress. Compared with conventional tissue culture seedlings, the survival rate of transplanting in mountainous areas is significantly improved, the seedling recovery period is greatly shortened, and it can quickly adapt to the harsh site conditions in northern mountainous areas.

[0016] 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

[0017] Figure 1 This is a schematic diagram of the relative growth rate of plant height in Examples 1-2 and Comparative Examples 1-2 of the present invention. Detailed Implementation

[0018] 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.

[0019] This invention provides a method for rapid propagation of apple rootstocks tolerant to poor soil through tissue culture, comprising the following steps: S1. Select mother trees that have been continuously planted on barren mountain land, select mother trees that are vigorous, virus-free and resistant to barrenness for scion collection, and cut stem segments with 2-5 plump axillary buds. S2. After cleaning the stem segments obtained in S1, soak them in a fungicide for 10-20 minutes, rinse them clean, and then perform low-temperature aging treatment. After cutting the stem segments that have undergone low-temperature aging treatment, soak them in ethanol, rinse them with sterile water, and then sterilize them in mercuric chloride solution. After rinsing with sterile water, use sterile paper to absorb the moisture, and cut off the browned wounds at both ends to obtain surface-sterilized explants. S3. Prepare an anti-browning bud germination medium. Place the surface-sterilized explants from S2 into the anti-browning bud germination medium and culture them at 24-25℃ for 20-30 days with a light duration of 15-16 h / d. S4. Prepare a subculture proliferation medium. Under aseptic conditions, divide the clustered shoots of the explants cultured in S3. Retain 2-3 effective buds in each clustered shoot. Inoculate the clustered shoots into the subculture proliferation medium and culture for 20-30 days to obtain sterile single seedlings. S5. Prepare a low-salt rooting medium, select sterile single seedlings obtained in S4, remove the aged callus at the base, inoculate them into the low-salt rooting medium, first culture in the dark for 3-7 days and then culture in the light for 15-20 days to obtain rooted seedlings. S6. Leave the culture bottle cap of S5 half-open for 2-5 days to gradually reduce humidity, close the stomata to adapt to the external environment, and prevent wilting due to water loss. Plant the rooted seedlings in a mixed substrate of peat moss and perlite for 12-18 days, then transplant them to a simulated poor soil substrate and continue to cultivate for 25-35 days to obtain rootstock seedlings with a height of 8-12cm.

[0020] In some embodiments of the present invention, in S1, the mother tree is continuously planted on barren mountain land for 2-3 years, and the scion harvesting time is in mid-April, with the stem segment length being 2-5 cm. The scion harvesting time is chosen when the mother tree has strong physiological activity, few endophytic bacteria, and plump axillary buds.

[0021] In some embodiments of the present invention, in step S2, the cleaning process involves: rinsing with running water for 60-90 minutes, using a soft brush to remove impurities and fuzz from the stem surface, soaking in a detergent containing a neutral surfactant, and then rinsing with running water. This cleaning removes dirt, fuzz, and some microorganisms from the stem surface, reducing the difficulty of subsequent sterilization.

[0022] In some embodiments of the present invention, in S2, the low-temperature juvenile treatment is as follows: the rinsed stem segments are wrapped in damp gauze and stored at 4°C in the dark for 6-8 days, with the length of the cut segments being 1-2 cm. Low temperature inhibits the activity of polyphenol oxidase, reducing the oxidation of phenolic substances; at the same time, it induces the stem segments to enter a physiological juvenile state, improving the axillary bud germination ability.

[0023] In some embodiments of the present invention, in step S2, the ethanol is 75% ethanol, the concentration of the mercuric chloride solution is 0.05-0.15%, and sterilization is performed in the mercuric chloride solution for 5-10 minutes. Ethanol provides rapid surface sterilization, while the mercuric chloride solution provides deep sterilization; their synergistic effect ensures a high sterilization rate.

[0024] In some embodiments of the present invention, in S3, the anti-browning bud germination medium consists of: MS, 0.8 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L ascorbic acid, 200 mg / L hydrolyzed casein, 0.5 g / L PVP, 28 g / L sucrose, and 6.5 g / L agar, with a pH of 5.6-5.8. 6-BA, in combination with NAA, promotes axillary bud germination while preventing callus formation. Ascorbic acid and PVP work synergistically to reduce or complex phenolic substances, completely inhibiting browning. Hydrolyzed casein provides amino acids and nitrogen, alleviating nutritional stress.

[0025] In some embodiments of the present invention, in S4, the subculture proliferation medium consists of: MS, 1.0 mg / L 6-BA, 0.1 mg / L IBA, 80 mg / L phloroglucinol, 20 mg / L adenine, 30 mg / L sodium thiosulfate, 25 g / L sucrose, and 6 g / L agar, with a pH of 5.6-5.8. Phloroglucinol promotes the separation between buds and increases the number of effective bud points; adenine promotes cell division and bud morphogenesis; and sodium thiosulfate continuously inhibits browning.

[0026] In some embodiments of the present invention, in S5, the low-salt rooting medium consists of: 1 / 4 MS, 1.0 mg / L IBA, 0.1 mg / L NAA, 0.3 g / L activated carbon, 60 mg / L betaine, 50 mg / L humic acid, 18 g / L sucrose, and 5.5 g / L agar, with a pH of 5.5-5.8. Activated carbon adsorbs root-inhibiting substances in agar and simultaneously creates a local dark environment, promoting root primordia differentiation. Betaine acts as an osmotic regulator, enhancing the seedlings' drought and barrenness tolerance; humic acid stimulates root cell elongation, improving root vitality.

[0027] In some embodiments of the present invention, in step S6, the mixing ratio of peat moss and perlite is 2-5:1, and 0.1-1% of slow-release organic fertilizer by mass of the mixed substrate is added to the mixed substrate. High porosity facilitates root aeration, and a small amount of slow-release fertilizer ensures early seedling growth without causing fertilizer damage.

[0028] In some embodiments of the present invention, in step S6, the mass ratio of garden soil, river sand, and weathered sandstone fragments in the infertile, thin soil matrix is ​​5-8:2-4:1, and a humic acid aqueous solution is sprayed weekly during the cultivation process. This simulates an infertile mountain soil environment. The weathered sandstone fragments provide trace mineral elements but do not retain water, forcing the roots to actively grow deeper and expand the absorption area. Weekly spraying of the humic acid aqueous solution continuously stimulates root development, improving the nutrient absorption efficiency of seedlings in an infertile environment.

[0029] Example 1 S1. Select mother trees that have been continuously planted on barren mountain land for 3 years. Select mother trees that are vigorous, virus-free and stable in barrenness for scion collection. The scion collection time is in early to mid-April. Cut stem segments with 2-3 plump axillary buds, and the length of the stem segments is 2-3cm.

[0030] S2. Wash the stem segments obtained in S1 with running water for 80 minutes. Use a soft brush to remove impurities and fuzz from the surface of the stem segments. Soak in a detergent containing a neutral surfactant and then rinse with running water. After washing, soak in a bactericide for 15 minutes, rinse thoroughly, wrap the rinsed stem segments in damp gauze, and store at 4°C in the dark for 8 days for low-temperature aging treatment. Then cut into segments of 1-2 cm in length. After cutting, soak in 75% ethanol for 40 seconds, rinse with sterile water, and then sterilize in 0.1% mercuric chloride solution for 7 minutes. After rinsing with sterile water, blot dry with sterile paper, and remove the browned wounds at both ends to obtain surface-sterilized explants.

[0031] S3. Prepare an anti-browning bud germination medium. The anti-browning bud germination medium consists of MS, 0.8 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L ascorbic acid (sterilized by filtration through a 0.22 μm membrane), 200 mg / L hydrolyzed casein, 0.5 g / L PVP, 28 g / L sucrose, and 6.5 g / L agar, with a pH of 5.6-5.8. Place the surface-sterilized explants from S2 into the anti-browning bud germination medium and incubate at 24°C for 25 days, with a photoperiod of 16 h / day.

[0032] S4. Prepare the subculture proliferation medium, which consists of MS, 1.0 mg / L 6-BA, 0.1 mg / L IBA, 80 mg / L phloroglucinol, 20 mg / L adenine, 30 mg / L sodium thiosulfate, 25 g / L sucrose, and 6 g / L agar, with a pH of 5.6-5.8. Under aseptic conditions, divide the clustered shoots of the explants cultured in S3, retaining 2-3 viable buds in each clustered shoot. Inoculate the clustered shoots into the subculture proliferation medium and culture for 25 days to obtain sterile single plantlets.

[0033] S5. Prepare a low-salt rooting medium consisting of: 1 / 4 MS, 1.0 mg / L IBA, 0.1 mg / L NAA, 0.3 g / L activated charcoal, 60 mg / L betaine, 50 mg / L humic acid, 18 g / L sucrose, and 5.5 g / L agar, with a pH of 5.5-5.8. Select sterile single seedlings obtained in S4, remove the aged callus at the base, and inoculate them into the low-salt rooting medium. Incubate in the dark for 5 days, followed by incubation in the light for 18 days to obtain rooted seedlings.

[0034] S6. Leave the culture bottles from S5 with the caps partially open for 4 days. Plant the rooted seedlings in a mixed substrate of peat moss and perlite for 15 days. The ratio of peat moss to perlite is 4:1. Add 1% slow-release organic fertilizer by weight of the mixed substrate to the substrate. After transplanting to a simulated poor soil substrate, continue cultivation for 28 days. The mass ratio of garden soil, river sand, and weathered sandstone debris in the poor soil substrate is 6:3:1. Spray humic acid solution weekly during the cultivation process to obtain rootstock seedlings with a height of 8-12cm.

[0035] Example 2 S1. Select mother trees that have been continuously planted on barren mountain land for 3 years. Select mother trees that are vigorous, virus-free and stable in barrenness for scion collection. The scion collection time is in early to mid-April. Cut stem segments with 2-3 plump axillary buds, and the length of the stem segments is 2-3cm.

[0036] S2. Wash the stem segments obtained in S1 with running water for 80 minutes. Use a soft brush to remove impurities and fuzz from the surface of the stem segments. Soak in a detergent containing a neutral surfactant and then rinse with running water. After washing, soak in a bactericide for 15 minutes, rinse thoroughly, wrap the rinsed stem segments in damp gauze, and store at 4°C in the dark for 6 days for low-temperature aging treatment. Then cut into segments of 1-2 cm in length. After cutting, soak in 75% ethanol for 40 seconds, rinse with sterile water, and then sterilize in 0.1% mercuric chloride solution for 7 minutes. After rinsing with sterile water, blot dry with sterile paper, and remove the browned wounds at both ends to obtain surface-sterilized explants.

[0037] S3. Prepare an anti-browning bud germination medium. The anti-browning bud germination medium consists of MS, 0.8 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L ascorbic acid (sterilized by filtration through a 0.22 μm membrane), 200 mg / L hydrolyzed casein, 0.5 g / L PVP, 28 g / L sucrose, and 6.5 g / L agar, with a pH of 5.6-5.8. Place the surface-sterilized explants from S2 into the anti-browning bud germination medium and incubate at 24°C for 23 days, with a photoperiod of 16 h / day.

[0038] S4. Prepare the subculture proliferation medium, which consists of MS, 1.0 mg / L 6-BA, 0.1 mg / L IBA, 80 mg / L phloroglucinol, 20 mg / L adenine, 30 mg / L sodium thiosulfate, 25 g / L sucrose, and 6 g / L agar, with a pH of 5.6-5.8. Under aseptic conditions, divide the clustered shoots of the explants cultured in S3, retaining 2-3 viable buds in each clustered shoot. Inoculate the clustered shoots into the subculture proliferation medium and culture for 28 days to obtain sterile single plantlets.

[0039] S5. Prepare a low-salt rooting medium consisting of: 1 / 4 MS, 1.0 mg / L IBA, 0.1 mg / L NAA, 0.3 g / L activated charcoal, 60 mg / L betaine, 50 mg / L humic acid, 18 g / L sucrose, and 5.5 g / L agar, with a pH of 5.5-5.8. Select sterile single seedlings obtained in S4, remove the aged callus at the base, and inoculate them into the low-salt rooting medium. Incubate in the dark for 4 days, followed by incubation in the light for 15 days to obtain rooted seedlings.

[0040] S6. Leave the culture bottles from S5 with the caps partially open for 4 days. Plant the rooted seedlings in a mixed substrate of peat moss and perlite for 15 days. The ratio of peat moss to perlite is 4:1. Add 1% slow-release organic fertilizer by weight of the mixed substrate to the substrate. After transplanting to a simulated poor soil substrate, continue cultivation for 28 days. The mass ratio of garden soil, river sand, and weathered sandstone debris in the poor soil substrate is 8:4:1. Spray humic acid solution weekly during the cultivation process to obtain rootstock seedlings with a height of 8-12cm.

[0041] Comparative Example 1 S1. Select mother trees that have been continuously planted on barren mountain land for 3 years. Select mother trees that are vigorous, virus-free and stable in barrenness for scion collection. The scion collection time is in early to mid-April. Cut stem segments with 2-3 plump axillary buds, and the length of the stem segments is 2-3cm.

[0042] S2. Wash the stem segments obtained in S1 with running water for 80 minutes. Use a soft brush to remove impurities and fuzz from the surface of the stem segments. Soak in a detergent containing a neutral surfactant and then rinse with running water. After washing, soak in a bactericide for 15 minutes. Cut the segments to a length of 1-2 cm. After cutting, soak in 75% ethanol for 40 seconds, rinse with sterile water, and then sterilize in 0.1% mercuric chloride solution for 7 minutes. After rinsing with sterile water, blot dry with sterile paper and remove the browned wounds at both ends to obtain surface-sterilized explants.

[0043] S3. Prepare an anti-browning bud germination medium. The anti-browning bud germination medium consists of MS, 0.8 mg / L 6-BA, 0.1 mg / L NAA, 0.5 g / L PVP, 28 g / L sucrose, and 6.5 g / L agar, with a pH of 5.6-5.8. Place the surface-sterilized explants from S2 into the anti-browning bud germination medium and culture at 24°C for 25 days, with a photoperiod of 16 h / day.

[0044] S4. Prepare the subculture proliferation medium, which consists of MS, 1.0 mg / L 6-BA, 0.1 mg / L IBA, 30 mg / L sodium thiosulfate, 25 g / L sucrose, and 6 g / L agar, with a pH of 5.6-5.8. Under aseptic conditions, divide the clustered shoots of the explants cultured in S3, retaining 2-3 viable buds in each clustered shoot. Inoculate the clustered shoots into the subculture proliferation medium and culture for 25 days to obtain sterile single seedlings.

[0045] S5. Prepare the rooting medium: MS medium with a pH of 5.5-5.8. Select sterile single seedlings obtained in S4, remove the aged callus at the base, inoculate them into the rooting medium, and culture them in the dark for 5 days followed by light culture for 18 days to obtain rooted seedlings.

[0046] S6. Leave the culture bottles from S5 with the caps partially open for 4 days. Plant the rooted seedlings in a mixed substrate of peat moss and perlite for 15 days. The ratio of peat moss to perlite is 4:1. Add 1% slow-release organic fertilizer by weight of the mixed substrate to the substrate. After transplanting to a simulated poor soil substrate, continue cultivation for 28 days. The mass ratio of garden soil, river sand, and weathered sandstone debris in the poor soil substrate is 6:3:1. Spray humic acid solution weekly during the cultivation process to obtain rootstock seedlings with a height of 8-12cm.

[0047] Comparative Example 2 S1. Select mother trees that have been continuously planted on barren mountain land for 3 years. Select mother trees that are vigorous, virus-free and stable in barrenness for scion collection. The scion collection time is in early to mid-April. Cut stem segments with 2-3 plump axillary buds, and the length of the stem segments is 2-3cm.

[0048] S2. Wash the stem segments obtained in S1 with running water for 80 minutes. Use a soft brush to remove impurities and fuzz from the surface of the stem segments. Soak in a detergent containing a neutral surfactant and then rinse with running water. After washing, soak in a bactericide for 15 minutes, rinse thoroughly, and then perform a low-temperature aging treatment. Wrap the washed stem segments in damp gauze and store at 4°C in the dark for 8 days. The length of the cut segments should be 1-2 cm. After cutting, soak in 75% ethanol for 40 seconds, rinse with sterile water, and then sterilize in 0.1% mercuric chloride solution for 7 minutes. After rinsing with sterile water, blot dry with sterile paper and remove the browned wounds at both ends to obtain surface-sterilized explants.

[0049] S3. Prepare an anti-browning bud germination medium. The anti-browning bud germination medium consists of MS, 0.8 mg / L 6-BA, 0.1 mg / L NAA, 0.5 g / L PVP, 28 g / L sucrose, and 6.5 g / L agar, with a pH of 5.6-5.8. Place the surface-sterilized explants from S2 into the anti-browning bud germination medium and culture at 24°C for 25 days, with a photoperiod of 16 h / day.

[0050] S4. Prepare the subculture proliferation medium, which consists of MS, 1.0 mg / L 6-BA, 0.1 mg / L IBA, 30 mg / L sodium thiosulfate, 25 g / L sucrose, and 6 g / L agar, with a pH of 5.6-5.8. Under aseptic conditions, divide the clustered shoots of the explants cultured in S3, retaining 2-3 viable buds in each clustered shoot. Inoculate the clustered shoots into the subculture proliferation medium and culture for 25 days to obtain sterile single seedlings.

[0051] S5. Prepare a low-salt rooting medium consisting of 1 / 4 MS, 1.0 mg / L IBA, 0.1 mg / L NAA, 50 mg / L humic acid, 18 g / L sucrose, and 5.5 g / L agar, with a pH of 5.5-5.8. Select sterile single seedlings obtained in S4, remove the aged callus at the base, and inoculate them into the low-salt rooting medium. Incubate in the dark for 5 days, followed by incubation in the light for 18 days to obtain rooted seedlings.

[0052] S6. Leave the culture bottle cap of S5 half open for 4 days, then plant the rooted seedlings in a mixed substrate of peat moss and perlite for 40 days. The mixing ratio of peat moss and perlite is 4:1. Add 1% slow-release organic fertilizer by weight of the mixed substrate to obtain rootstock seedlings with a height of 8-12cm.

[0053] Performance testing The seedlings of Examples 1-2 and Comparative Examples 1-2 were dug up 30 days after transplanting, and the complete root system was examined. The root volume, number of root tips and root vigor were tested. The results are shown in Table 1.

[0054] Table 1. Root system data for Examples 1-2 and Comparative Examples 1-2

[0055] The rootstock seedlings obtained in Examples 1-2 and Comparative Examples 1-2 were transplanted into a low-nitrogen substrate (river sand:perlite mass ratio 3:1) and watered with 1 / 10 Hoagland nutrient solution (nitrogen deficiency). After 21 days of cultivation, the relative growth rate of plant height, chlorophyll content, and free proline content were measured. The results are shown in Table 2 and... Figure 1 As shown.

[0056] Table 2. Low nitrogen stress test results of Examples 1-2 and Comparative Examples 1-2

[0057] The rootstock seedlings obtained from Examples 1-2 and Comparative Examples 1-2 were transplanted to barren mountain land. One year after planting, the leaf nutrient content, trunk diameter and disease incidence were observed. The results are shown in Table 3.

[0058] Table 3. Data after transplanting in Examples 1-2 and Comparative Examples 1-2

[0059] As shown in Tables 1-3, compared with Comparative Examples 1-2, the rootstock seedlings cultivated in Examples 1-2 exhibited superior root growth and tolerance to poor soil conditions. Under low nitrogen stress, the tolerant plants mitigated nitrogen deficiency-induced metabolic disorders by accumulating large amounts of free proline as an osmotic regulator and antioxidant. Robust root systems and balanced nutrient supply enhanced the overall disease resistance of the plants, enabling them to maintain relatively normal nutrient levels and growth rates in barren mountain environments.

[0060] Therefore, this invention adopts the above-mentioned method for rapid propagation of apple rootstocks tolerant to poor soil conditions through tissue culture. It selects robust, virus-free mother trees that have been continuously planted in poor mountainous areas of northern China and have stable traits of resistance to poor soil conditions for scion collection. Through low-temperature juvenile treatment, it effectively enhances the activity and stress resistance of explants. Using two substrates, the seedlings are gradually adapted to the poor, well-aerated, and nutrient-deficient mountainous soil environment, which greatly improves the seedlings' resistance to poor soil conditions and stress resistance.

[0061] 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 apple rootstocks tolerant of poor soil through tissue culture, characterized in that: Includes the following steps: S1. Select mother trees that have been continuously planted on barren mountain land, select mother trees that are vigorous, virus-free and resistant to barrenness for scion collection, and cut stem segments with 2-5 plump axillary buds. S2. After cleaning the stem segments obtained in S1, soak them in a fungicide for 10-20 minutes, rinse them clean, and then perform low-temperature aging treatment. After cutting the stem segments that have undergone low-temperature aging treatment, soak them in ethanol, rinse them with sterile water, and then sterilize them in mercuric chloride solution. After rinsing with sterile water, use sterile paper to absorb the moisture, and cut off the browned wounds at both ends to obtain surface-sterilized explants. S3. Prepare an anti-browning bud germination medium. Place the surface-sterilized explants from S2 into the anti-browning bud germination medium and culture them at 24-25℃ for 20-30 days with a light duration of 15-16 h / d. S4. Prepare a subculture proliferation medium. Under aseptic conditions, divide the clustered shoots of the explants cultured in S3. Retain 2-3 effective buds in each clustered shoot. Inoculate the clustered shoots into the subculture proliferation medium and culture for 20-30 days to obtain sterile single seedlings. S5. Prepare a low-salt rooting medium, select sterile single seedlings obtained in S4, remove the aged callus at the base, inoculate them into the low-salt rooting medium, first culture in the dark for 3-7 days and then culture in the light for 15-20 days to obtain rooted seedlings. S6. Leave the culture bottle cap in S5 half open for 2-5 days. Plant the rooted seedlings in a mixed substrate of peat moss and perlite for 12-18 days. After transplanting to a simulated barren soil substrate, continue to cultivate for 25-35 days to obtain rootstock seedlings with a plant height of 8-12cm.

2. The method for rapid propagation of apple rootstocks tolerant to poor soil by tissue culture according to claim 1, characterized in that: In S1, the mother tree is continuously planted on barren mountain land for 2-3 years, and the scion time is in mid-April, with the stem segment length being 2-5cm.

3. The method for rapid propagation of apple rootstocks tolerant to poor soil by tissue culture according to claim 1, characterized in that: In S2, the cleaning process is as follows: wash with running water for 60-90 minutes, use a soft brush to remove impurities and fuzz from the surface of the stem segments, soak in a detergent containing a neutral surfactant, and then rinse with running water.

4. The method for rapid propagation of apple rootstocks tolerant to poor soil by tissue culture according to claim 1, characterized in that: In S2, the low-temperature aging treatment is as follows: the rinsed stem segments are wrapped in damp gauze and stored at 4°C in the dark for 6-8 days, and the length of the cut segments is 1-2 cm.

5. The method for rapid propagation of apple rootstocks tolerant to poor soil by tissue culture according to claim 1, characterized in that: In S2, the ethanol is 75% ethanol, the concentration of mercuric chloride solution is 0.05-0.15%, and sterilization is carried out in mercuric chloride solution for 5-10 minutes.

6. The method for rapid propagation of apple rootstocks tolerant to poor soil by tissue culture according to claim 1, characterized in that: In S3, the anti-browning bud germination medium consisted of MS, 0.8 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L ascorbic acid, 200 mg / L hydrolyzed casein, 0.5 g / L PVP, 28 g / L sucrose, and 6.5 g / L agar, with a pH of 5.6-5.

8.

7. The method for rapid propagation of apple rootstocks tolerant to poor soil by tissue culture according to claim 1, characterized in that: In S4, the subculture culture medium consisted of MS, 1.0 mg / L 6-BA, 0.1 mg / L IBA, 80 mg / L phloroglucinol, 20 mg / L adenine, 30 mg / L sodium thiosulfate, 25 g / L sucrose, and 6 g / L agar, with a pH of 5.6-5.

8.

8. The method for rapid propagation of apple rootstocks tolerant to poor soil by tissue culture according to claim 1, characterized in that: In S5, the low-salt rooting medium consisted of 1 / 4 MS, 1.0 mg / L IBA, 0.1 mg / L NAA, 0.3 g / L activated carbon, 60 mg / L betaine, 50 mg / L humic acid, 18 g / L sucrose, and 5.5 g / L agar, with a pH of 5.5-5.

8.

9. The method for rapid propagation of apple rootstocks tolerant to poor soil by tissue culture according to claim 1, characterized in that: In S6, the mixing ratio of peat moss and perlite is 2-5:1, and slow-release organic fertilizer accounting for 0.1-1% of the mass of the mixed substrate is added to the mixed substrate.

10. The method for rapid propagation of apple rootstocks tolerant to poor soil by tissue culture according to claim 1, characterized in that: In S6, the mass ratio of garden soil, river sand and weathered sandstone debris in the barren thin soil matrix is ​​5-8:2-4:1, and humic acid aqueous solution is sprayed weekly during the cultivation process.

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