Method for creating new germplasm by grafting potato tissue culture seedlings

Abstract

The invention discloses a method for creating new germplasm by grafting potato tissue culture seedlings, relates to the technical field of potato breeding, and solves the problems of long creation period, high parent selection difficulty and high screening cost of traditional potato new germplasm in cross breeding, mutation breeding and the like. Comprising the steps of parent tissue culture seedling cultivation, pretreatment before grafting, tissue culture seedling grafting, grafted seedling callus culture, seedling hardening domestication and germplasm screening. The method shortens the stable period of the new germplasm character, is not limited by seasons, multiplies the breeding efficiency, realizes efficient creation of the new potato germplasm, and can improve the breeding survival rate and the germplasm creation efficiency.

Description

A method for creating new germplasm by grafting potato tissue culture seedlings Technical Field

[0001] This invention relates to the field of potato breeding technology, and specifically to a method for creating new germplasm by grafting potato tissue culture seedlings. Background Technology

[0002] As one of the world's most important food crops, potatoes play an irreplaceable role in ensuring food security and promoting agricultural economic development. Excellent germplasm resources are the core foundation for potato variety improvement and healthy industrial development. At present, the creation of new potato germplasm mainly relies on traditional techniques such as hybridization breeding and mutation breeding, but these methods have some significant limitations: (1) Hybridization breeding has a long cycle and low efficiency: Traditional hybridization breeding usually takes 5-8 years from parent selection to obtaining a stable new line, which is a long cycle and difficult to meet the timeliness requirements of modern breeding. At the same time, potatoes generally have self-incompatibility and inbreeding depression, which leads to a limited range of parent selection and low hybridization fruit set rate, further increasing the difficulty and uncertainty of breeding.

[0003] (2) Mutation breeding has uncontrollable variation and high screening costs: Although physical or chemical mutagenesis can expand the range of genetic variation, the direction of variation is random, the frequency of beneficial variation is extremely low (usually less than 0.1%), and the vast majority of variation is unfavorable trait. In order to screen out a very small number of target phenotypes, it is necessary to construct a large-scale population and conduct field identification in multiple locations over many years, which consumes a lot of manpower, material resources and land resources, thus increasing the cost of quantitative screening.

[0004] With the continuous development of plant tissue culture technology, potato tissue culture seedlings, due to their advantages such as uniform growth, consistent genetic background, and absence of pathogen contamination, have been widely used for the rapid propagation of virus-free potato seed tubers. However, research on the systematic application of tissue culture seedling grafting technology to potato germplasm creation is still in its early stages, and no mature, publicly available technical solutions have yet been discovered.

[0005] Therefore, to address the pain points of traditional breeding techniques, such as long cycles, low efficiency, and high costs, there is an urgent need to develop a novel germplasm creation method based on grafting of tissue culture seedlings. This method should possess advantages such as ease of operation, high grafting survival rate, and a clear genetic fusion mechanism, effectively overcoming the inherent defects of hybridization and mutation breeding, and providing a new technical pathway for potato germplasm resource innovation.

[0006] Therefore, there is an urgent need to develop a simple, high-survival-rate, and efficient method for grafting potato tissue culture seedlings to address the shortcomings of traditional germplasm creation techniques. Summary of the Invention

[0007] To address the above shortcomings, this invention provides a method for creating new germplasm by grafting potato tissue culture seedlings, which shortens the stabilization cycle of new germplasm traits and is not limited by season, thus doubling breeding efficiency and achieving efficient creation of new potato germplasm. This solves the problems of long creation cycle, difficult parent selection, and high screening cost in traditional potato germplasm creation methods such as hybridization breeding and mutation breeding, and can improve breeding survival rate and germplasm creation efficiency. The specific technical solution is as follows: The purpose of this invention is to provide a method for creating new germplasm by grafting potato tissue culture seedlings, including the following steps: (1) Parent tissue culture seedling cultivation: Select potato varieties with target traits as rootstocks and potato varieties with ordinary traits as scions. Take disease-free stem tips as explants, disinfect the explants, and inoculate them into MS basal medium for cultivation to obtain scion seedlings and rootstock seedlings respectively; (2) Pre-grafting treatment: Harden the scion seedlings and rootstock seedlings, cut off the terminal bud of the rootstock seedlings, cut the rootstock seedlings into stem segments, remove the leaves, lightly apply the grafting pretreatment solution to the cut of the rootstock seedlings and the base of the scion, let it stand, and then insert the end near the root into the tissue culture seedling culture medium for cultivation to obtain the pre-treated scion seedlings and rootstock seedlings; (3) grafting of tissue culture seedlings: graft the base of the scion into the cut of the rootstock, and tightly wrap the grafting joint with biodegradable glutinous rice paper grafting film; (4) callus culture of grafted seedlings: inoculate the grafted seedlings into the callus culture medium for grafted seedling callus culture; (5) hardening and germplasm screening: transfer the grafted seedlings to the hardening culture medium for hardening, and then transplant the grafted seedlings into sterile substrate soil, and manage them in the greenhouse until the potato tubers mature, harvest the potato seed tubers, and obtain the newly created potato germplasm.

[0008] Preferably, in step (1), the explant is 2-3 cm long and has 1-2 axillary buds; the target trait is one or more of the following: resistance, high starch content, and high protein content.

[0009] Preferably, in step (1), the disinfection is first soaked in 75% ethanol for 30-45 seconds, then disinfected with 0.1% mercuric chloride solution for 5-6 minutes, and then rinsed with sterile water 4-5 times.

[0010] Preferably, in step (1), the MS basal medium is supplemented with 30 g / L sucrose and 7 g / L agar, and the pH is 5.9; the culture is carried out in a culture room with conditions of 20±1℃, light intensity of 2000~2500 lux, and light duration of 14h / d for 20~30 days; the scion seedlings and rootstock seedlings are robust tissue culture seedlings with a plant height of 8~10cm and a stem diameter of 0.1~0.2cm.

[0011] Preferably, in step (2), the hardening-off pretreatment involves transferring the scion seedlings and rootstock seedlings to a culture environment with a light intensity of 3000 lux 3 days before grafting; the rootstock seedlings are cut into stem segments of 2.8–3.2 cm in length before grafting. The hardening-off pretreatment before grafting enhances the stress resistance of the tissue culture seedlings; at the same time, the terminal buds of the rootstock seedlings are removed, leaving a 4–5 cm stem segment at the base, which promotes the inhibition of lateral bud germination and reduces nutrient consumption.

[0012] Preferably, in step (2), the grafting pretreatment solution is prepared by dissolving 20 mg / L naphthaleneacetic acid (NAA) and 10 mg / L 6-benzylaminopurine (6-BA) in sterile water and stirring until homogeneous; the standing time is 10-15 min. A sterile cotton swab is used to apply the grafting pretreatment solution lightly to the rootstock incision (the subsequent grafting incision) and the base of the scion, and the solution is left to stand to promote callus formation.

[0013] Preferably, in step (2), the tissue culture medium is made from the following raw materials in parts by weight: 4.2-4.7 parts MS medium, 28-33 parts sucrose, 0.05-0.15 parts inositol, 6-8 parts agar, 0.1-0.2 parts vitamin B1, 0.1-0.3 parts vitamin C, 0.1-0.2 parts vitamin B6, 0.8-1.3 parts 0.5-2 mg / L 6-benzylaminopurine, and 0.05-0.2 parts 0.1-0.5 mg / L naphthaleneacetic acid. The materials are mixed evenly, the pH is adjusted to 5.6-5.8, and sterilized to obtain the final product.

[0014] Preferably, in step (3), the grafting adopts the "cleft grafting method," the specific steps of which are as follows: On a sterile operating table, a sterile scalpel is used to cleft the top of the rootstock stem segment downward along the central axis, with a cut depth of 1.5-2 cm and a cut width of 0.1-0.2 cm. The cut width matches the diameter of the scion base. The base of the scion is cut into a wedge shape with a length of 1.5-2 cm, and the scion base is inserted into the cut of the rootstock to complete the grafting; the wrapping is done by wrapping 2-3 times. The length of the wedge shape at the base of the scion is consistent with the depth of the rootstock cut to ensure that the cambium layers of the scion and the rootstock are aligned. The tightness of the wrapping should be such that it does not damage the stem segment and there are no gaps, avoiding moisture loss and microbial contamination.

[0015] Preferably, the biodegradable glutinous rice paper grafting film is a commercially available product that can be purchased on the market.

[0016] Preferably, in step (4), the callus culture medium is MS basal medium + 15 g / L sucrose + 7 g / L agar + 10 mg / L NAA + 5 mg / L 6-BA, with a pH of 5.8.

[0017] Preferably, in step (4), the grafted seedling callus culture is as follows: During the initial 0-7 days of callus culture, the culture conditions are 25±1℃ and a dark environment; after 7 days, the conditions are adjusted to 23±2℃, light intensity 2000 lux, and light duration 14h / d; during the culture period, the grafted seedling status is observed every 5 days. If there is condensation inside the biodegradable glutinous rice paper grafting film, it is promptly dried with a sterile cotton swab. The initial callus culture promotes rapid callus formation at the graft union; after 7 days, the culture conditions are adjusted to induce callus differentiation into vascular tissue; during the grafted seedling callus culture, the condensation inside the biodegradable glutinous rice paper grafting film is promptly dried with a sterile cotton swab to prevent bacterial growth.

[0018] Preferably, in step (5), the hardening-off medium is MS basal medium + 20 g / L sucrose + 7 g / L agar, with a pH of 5.8; the hardening-off time is 5-7 days, under the following conditions: temperature 20-25℃, humidity 70%-80%, and natural diffused light; the sterile substrate soil is peat moss and perlite in a mass ratio of 3:1; the temperature of the greenhouse under normal management is 22-25℃, and the humidity is maintained at 60%-70%. During hardening-off, the cap of the culture bottle containing the hardening-off medium is opened, leaving only a layer of sterile gauze, and the bottle is placed in the greenhouse to allow the grafted seedlings to adapt to the external environment.

[0019] The present invention achieves at least the following beneficial effects: 1. By optimizing key steps such as tissue culture seedling cultivation, grafting operation, callus culture and seedling hardening and domestication, the present invention shortens the stabilization cycle of new germplasm traits, is not limited by season, doubles the breeding efficiency, and realizes the efficient creation of new germplasm.

[0020] 2. This invention, through asexual grafting, bypasses sexual reproduction barriers such as self-incompatibility and inbreeding depression in potatoes, enabling trait fusion between parental materials that are difficult to achieve gene exchange through hybridization. This opens up new avenues for utilizing superior genes from special germplasm resources such as wild species and local varieties. By utilizing the low-frequency variations that may spontaneously arise in plant somatic cells during tissue culture and grafting healing, additional genetic diversity is added to the creation of new germplasm, potentially yielding new traits that are difficult to achieve through sexual hybridization, thus broadening the genetic basis of potato breeding.

[0021] 3. Unlike the random and uncontrollable mutations in mutation breeding, this invention purposefully selects tissue culture seedlings with specific traits (such as high resistance and excellent quality) as rootstocks or scions, making the trait variation of newly created germplasm more directional and predictable, effectively avoiding the generation of a large number of invalid mutations in mutation breeding, thereby significantly reducing the scale and cost of later screening; grafting can quickly express new traits generated by the interaction between rootstock and scion on the grafted body, providing an efficient technical means for rapidly obtaining potatoes with specific traits. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 shows the tissue culture seedlings prepared for grafting according to the present invention; Figure 2 shows the rootstock seedling stem segments used for propagation into tissue culture seedlings according to the present invention; Figure 3 shows the growth of the rootstock seedling stem segments on the first day of cultivation according to the present invention; Figure 4 shows the growth of the rootstock seedling stem segments on the fifth day of cultivation according to the present invention; Figure 5 shows the growth of the rootstock seedling stem segments on the seventh day of cultivation according to the present invention; Figure 6 shows the growth of the rootstock seedling stem segments on the eleventh day of cultivation according to the present invention. Detailed Implementation

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0025] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0026] Potatoes rarely flower and bear fruit in Guangxi, and conventional breeding is very difficult. Therefore, if you want a better potato variety (such as a cold-resistant variety), you can only introduce varieties from other provinces, or improve the quality of the original varieties (such as improving cold resistance) through grafting. However, ordinary people cannot perform grafting operations.

[0027] Traditional methods for creating new potato germplasm mainly rely on hybridization breeding and mutation breeding. However, the inventors of this application have discovered significant limitations in this approach through long-term research: hybridization breeding has a long cycle (usually 5-8 years) and is difficult to pair with parents due to potato self-incompatibility; mutation breeding results in uncontrollable variation directions, low beneficial variation rates (usually less than 0.1%), and high screening costs. Furthermore, traditional grafting requires a one-to-one pair of rootstock and scion, leading to a large demand for seed potatoes.

[0028] The inventors discovered through in-depth research on tissue culture technology that potato tissue culture seedlings have many advantages such as uniform growth and no virus contamination, making them very suitable for the rapid propagation of potato seed tubers. However, research on using tissue culture technology for grafting to create new germplasm is still in its early stages, and no relevant literature has been published.

[0029] Therefore, in order to address the shortcomings of traditional germplasm creation techniques, the inventors conducted further in-depth research on tissue culture technology and ultimately developed a simple, high-survival-rate, and highly efficient method for grafting potato tissue culture seedlings, as detailed below.

[0030] A method for creating new germplasm by grafting potato tissue culture seedlings includes the following steps: (1) Cultivation of parent tissue culture seedlings: Select potato varieties with target traits (such as resistance, high starch content, high protein content, etc.) as rootstocks, and potato varieties with ordinary traits as scions. Take disease-free stem tips with a length of 2-3 cm and 1-2 axillary buds as explants. Soak the explants in 75% ethanol for 30-45 seconds, then disinfect them with 0.1% mercuric chloride solution for 5-6 minutes, and then use... After rinsing with sterile water 4-5 times, the tissue culture seedlings were inoculated into MS basal medium (with 30 g / L sucrose, 7 g / L agar, and pH adjusted to 5.9) and placed in a culture room with culture conditions of 20±1℃, light intensity of 2000-2500 lux, and light duration of 14 h / d for 20-30 days to obtain robust tissue culture seedlings with a plant height of 8-10 cm and a stem diameter of 0.1-0.2 cm. Scion seedlings and rootstock seedlings were obtained respectively; (2) Pretreatment before grafting: 3 days before grafting, Both scion and rootstock seedlings were transferred to a cultivation environment with a light intensity of 3000 lux for hardening-off pretreatment to enhance the stress resistance of the tissue culture seedlings. Simultaneously, the terminal buds of the rootstock seedlings were removed, and the seedlings were cut into 2.8–3.2 cm long stem segments. All leaves were removed to inhibit the germination of lateral buds and reduce nutrient consumption. A grafting pretreatment solution was prepared by dissolving 20 mg / L naphthaleneacetic acid (NAA) and 10 mg / L 6-benzylaminopurine (6-BA) in sterile water and stirring thoroughly. Dip a cotton swab in the grafting pretreatment solution and lightly apply it to the incision site on the rootstock (the site of the subsequent grafting) and the base of the scion. Let it stand for 10-15 minutes, then insert the end closest to the root into the tissue culture medium for cultivation to promote callus formation. The tissue culture medium is prepared from the following ingredients by weight: MS medium 4.2-4.7 parts, sucrose 28-33 parts, inositol 0.05-0.15 parts, agar 6-8 parts, vitamin B1 0.1-0.2 parts, and vitamin C. 0.1-0.3 parts, vitamin B6 0.1-0.2 parts, 0.5-2 mg / L 6-benzylaminopurine 0.8-1.3 parts, 0.1-0.5 mg / L naphthaleneacetic acid 0.05-0.2 parts, mix the materials evenly, adjust the pH value to 5.6-5.8, and sterilize to obtain the product; (3) grafting operation of tissue culture seedlings: grafting is carried out by cleft grafting, specifically: on a sterile operating table, use a sterile scalpel to cleft the top of the rootstock stem segment downward along the central axis, the cut depth is 1.5-2cm, and the cut width matches the diameter of the scion base (0.1-0.2cm); cut the base of the scion into a wedge shape (the length is consistent with the cut depth of the rootstock, 1.(5-2cm), ensuring the cambium layers of the scion and rootstock are aligned; then tightly wrap the graft union with biodegradable glutinous rice paper grafting film (wrapping 2-3 times, the tightness should be such that it does not damage the stem segment and there are no gaps), to avoid water loss and microbial contamination; (4) Grafted seedling callus culture: immediately inoculate the grafted seedlings into callus culture medium (MS basal medium + 15g / L sucrose + 7g / L agar + 10mg / L NAA + 5mg / L 6-BA, pH adjusted to 5.8). In the early stage of callus culture (0-7 days), set the culture conditions to 25±1℃ and dark environment to promote the rapid formation of callus tissue at the graft union; after 7 days, adjust to 23±2℃, light intensity 2000lux, light time 14h / d environment to induce callus tissue to differentiate into vascular tissue; during the grafted seedling callus culture, use sterile cotton swabs to dry the condensate water generated in the biodegradable glutinous rice paper grafting film in time to avoid the growth of pathogens.

[0031] (5) Hardening and germplasm screening: Transfer the grafted seedlings to the “hardening medium” (MS basal medium + 20 g / L sucrose + 7 g / L agar, pH adjusted to 5.8), and open the bottle cap (keeping a layer of sterile gauze), place them in the greenhouse (temperature 20-25℃, humidity 70%-80%, natural diffused light) for hardening for 5-7 days to allow the grafted seedlings to adapt to the external environment; after hardening, transplant the grafted seedlings into sterile substrate soil (peat soil: perlite = 3:1), and manage the greenhouse in a conventional manner (maintain humidity 60%-70%, temperature 22-25℃), and the resulting seed potatoes are the newly created potato germplasm.

[0032] Example 1 A method for creating new germplasm by grafting potato tissue culture seedlings includes the following steps: (1) Cultivation of parent tissue culture seedlings: Select potato varieties with target traits (resistance, high starch content) as rootstocks and potato varieties with ordinary traits as scions. Take disease-free stem tips with a length of 2cm and one axillary bud as explants. First, soak the explants in 75% ethanol for 30s, then disinfect them with 0.1% mercuric chloride solution for 5min, then rinse them with sterile water 4 times, and then inoculate them into MS basal medium (added with 30g / L sucrose, 7g / L agar, pH 5.9). Cultivate them in a culture room at 20±1℃, light intensity of 2000 lux, and light duration of 14h / d for 20d to obtain robust tissue culture seedlings with a plant height of 8cm and a stem diameter of 0.1cm. Obtain scion seedlings and rootstock seedlings respectively; (2) Pre-grafting treatment: Three days before grafting, the scion and rootstock seedlings were transferred to a cultivation environment with a light intensity of 3000 lux for hardening-off pre-treatment. The terminal bud of the rootstock seedling was removed, and the rootstock seedling was cut into 2.8 cm long stem segments. After removing all leaves, the grafting pre-treatment solution (prepared by dissolving 20 mg / L naphthaleneacetic acid (NAA) and 10 mg / L 6-benzylaminopurine (6-BA) in sterile water and stirring evenly) was lightly applied to the cut of the rootstock seedling and the base of the scion. After standing for 10 minutes, the end closest to the root was inserted into the tissue culture medium for cultivation to obtain pre-treated scion and rootstock seedlings. The tissue culture medium was prepared from the following raw materials in parts by weight: MS medium 4.2 parts, sucrose 28 parts, inositol 0.05 parts, agar 6 parts, vitamin B1 0.1 parts, vitamin C 0.1 parts. 0.1 parts, vitamin B6 0.1 parts, 0.8 parts of 0.5 mg / L 6-benzylaminopurine, 0.05 parts of 0.1 mg / L naphthaleneacetic acid, mix the materials evenly, adjust the pH value to 5.6, and sterilize to obtain the product; (3) grafting of tissue culture seedlings: grafting is carried out by "cleft grafting". The specific steps are as follows: on a sterile operating table, use a sterile scalpel to chop the top of the rootstock stem segment downward along the central axis. The cut depth is 1.5 cm and the cut width is 0.1 cm. The cut width matches the diameter of the scion base. Cut the scion base into a wedge shape with a length of 1.5 cm. Insert the scion base into the rootstock cut. Then wrap the graft union tightly with biodegradable glutinous rice paper grafting film for 2 turns; (4) callus culture of grafted seedlings: inoculate the grafted seedlings into callus culture medium (MS basal medium + 15 g / L sucrose + 7 g / L agar + 10 mg / L NAA + 5 mg / L 6-BA, pH 5).8) Callus culture of grafted seedlings was carried out. In the early stage of callus culture, the culture conditions were 25±1℃ and dark environment for 0-7 days. After 7 days, the temperature was adjusted to 23±2℃, light intensity of 2000 lux and light duration of 14h / d. During the culture period, the condition of grafted seedlings was observed every 5 days. If there was condensation in the grafting film, it was dried with sterile cotton swabs in time. (5) Seedling hardening and germplasm screening: The grafted seedlings were transferred to the seedling hardening medium (MS basal medium + 20g / L sucrose + 7g / L agar, pH 5.8) for 5 days of hardening. The hardening temperature was 20℃, the humidity was 70% and natural diffused light was used. Then the grafted seedlings were transplanted into sterile substrate soil (peat soil: perlite = 3:1) and managed in the greenhouse (temperature 22℃, humidity 60%) until the potato tubers matured. The potato seed tubers were harvested to obtain the newly created potato germplasm.

[0033] Example 2 A method for creating new germplasm by grafting potato tissue culture seedlings, including the following steps: (1) Cultivation of parent tissue culture seedlings: Select potato varieties with target traits (high starch content, high protein content) as rootstocks, and select potato varieties with ordinary traits as scions. Take disease-free stem tips with a length of 3cm and 2 axillary buds as explants. First, soak the explants in 75% ethanol for 45s, then disinfect them with 0.1% mercuric chloride solution for 6min, then rinse them 5 times with sterile water, and then inoculate them into MS basal medium (added with 30g / L sucrose, 7g / L agar, pH 5.9). Cultivate them in a culture room at 20±1℃, light intensity of 2500 lux, and light duration of 14h / d for 30d to obtain robust tissue culture seedlings with a plant height of 10cm and a stem diameter of 0.2cm. Obtain scion seedlings and rootstock seedlings respectively; ( 2) Pre-grafting treatment: Three days before grafting, the scion and rootstock seedlings were transferred to a cultivation environment with a light intensity of 3000 lux for hardening-off pre-treatment. The terminal bud of the rootstock seedling was removed, and the rootstock seedling was cut into 3.2 cm long stem segments. After removing all leaves, the grafting pre-treatment solution (prepared by dissolving 20 mg / L naphthaleneacetic acid (NAA) and 10 mg / L 6-benzylaminopurine (6-BA) in sterile water and stirring evenly) was lightly applied to the cut of the rootstock seedling and the base of the scion. After standing for 15 minutes, the end closest to the root was inserted into the tissue culture medium for cultivation to obtain pre-treated scion and rootstock seedlings. The tissue culture medium was prepared from the following raw materials in parts by weight: MS medium 4.7 parts, sucrose 33 parts, inositol 0.15 parts, agar 8 parts, vitamin B1 0.2 parts, vitamin C 0.2 parts. 0.3 parts, vitamin B6 0.2 parts, 2mg / L 6-benzylaminopurine 1.3 parts, 0.5mg / L naphthaleneacetic acid 0.2 parts, mix the materials evenly, adjust the pH value to 5.8, and sterilize to obtain the product; (3) grafting of tissue culture seedlings: grafting is carried out by "cleft grafting". The specific steps are as follows: on the sterile operating table, use a sterile scalpel to chop the top of the rootstock stem segment downward along the central axis. The cut depth is 2cm, the cut width is 0.1cm, and the cut width matches the diameter of the scion base. Cut the scion base into a wedge shape with a length of 2cm, insert the scion base into the rootstock cut, and then use biodegradable glutinous rice paper grafting film to tightly wrap the graft union 3 times; (4) callus culture of grafted seedlings: inoculate the grafted seedlings into callus culture medium (MS basal medium + 15g / L sucrose + 7g / L agar + 10mg / L NAA + 5mg / L 6-BA, pH 5).8) Callus culture of grafted seedlings was carried out. In the early stage of callus culture, the culture conditions were 25±1℃ and dark environment for 0-7 days. After 7 days, the temperature was adjusted to 23±2℃, light intensity of 2000 lux and light duration of 14h / d. During the culture period, the condition of grafted seedlings was observed every 5 days. If there was condensation in the grafting film, it was dried with sterile cotton swabs in time. (5) Seedling hardening and germplasm screening: The grafted seedlings were transferred to the seedling hardening medium (MS basal medium + 20g / L sucrose + 7g / L agar, pH 5.8) for 7 days of hardening. The hardening temperature was 25℃, the humidity was 80%, and natural diffused light was used. Then the grafted seedlings were transplanted into sterile substrate soil (peat soil: perlite = 3:1) and managed in the greenhouse (temperature 25℃, humidity maintained at 70%) until the potato tubers matured. The potato seed tubers were harvested to obtain the newly created potato germplasm.

[0034] Example 3 A method for creating new germplasm by grafting potato tissue culture seedlings includes the following steps: (1) Cultivation of parent tissue culture seedlings: Select potato varieties with target traits (one or more of resistance, high starch content, and high protein content) as rootstocks, and select potato varieties with ordinary traits as scions. Take disease-free stem tips with a length of 2.5 cm and 1.5 axillary buds as explants. First, soak the explants in 75% ethanol for 38 seconds, then disinfect them with 0.1% mercuric chloride solution for 5.5 minutes, and then rinse them 5 times with sterile water. Then, inoculate them into MS basal medium (add 30 g / L sucrose, 7 g / L agar, pH 5.9) and culture them in a culture room at 20±1℃, light intensity of 2300 lux, and light duration of 14 h / d for 25 days to obtain robust tissue culture seedlings with a plant height of 9 cm and a stem diameter of 0.15 cm. Scion seedlings and rootstock seedlings; (2) Pre-treatment before grafting: 3 days before grafting, the scion seedlings and rootstock seedlings are transferred to a culture environment with a light intensity of 3000 lux for hardening pretreatment. The terminal bud of the rootstock seedling is cut off, and the rootstock seedling is cut into 3.0 cm long stem segments. After removing all leaves, the grafting pretreatment solution (preparation method: dissolve 20 mg / L naphthaleneacetic acid (NAA) and 10 mg / L 6-benzylaminopurine (6-BA) in sterile water and stir evenly) is lightly applied to the cut of the rootstock seedling and the base of the scion. After standing for 13 min, the end near the root is inserted into the tissue culture medium for culture to obtain the pretreated scion seedlings and rootstock seedlings; wherein, the tissue culture medium is made from the following raw materials in parts by weight: MS medium 4.5 parts, sucrose 30 parts, inositol 0.1 parts, agar 7 parts, vitamin B1 0.15 parts, vitamin C 0.2 parts, vitamin B6 0.15 parts, 1.0 part of 1.2 mg / L 6-benzylaminopurine, 0.1 part of 0.3 mg / L naphthaleneacetic acid, mix the materials evenly, adjust the pH value to 5.7, and sterilize to obtain the product; (3) grafting of tissue culture seedlings: grafting is carried out by "cleft grafting". The specific steps are as follows: on a sterile operating table, use a sterile scalpel to chop the top of the rootstock stem segment downward along the central axis. The cut depth is 1.8 cm and the cut width is 0.15 cm. The cut width matches the diameter of the scion base. Cut the scion base into a wedge shape with a length of 1.8 cm. Insert the scion base into the rootstock cut. Then wrap the grafting junction tightly with biodegradable glutinous rice paper grafting film for 3 turns; (4) callus culture of grafted seedlings: inoculate the grafted seedlings into callus culture medium (MS basal medium + 15 g / L sucrose + 7 g / L agar + 10 mg / L NAA + 5 mg / L 6-BA, pH 5.8) Callus culture of grafted seedlings was carried out. In the early stage of callus culture, the culture conditions were 25±1℃ and dark environment for 0-7 days. After 7 days, the temperature was adjusted to 23±2℃, light intensity of 2000 lux and light time of 14h / d. During the culture period, the condition of grafted seedlings was observed every 5 days. If there was condensation in the grafting film, it was dried with sterile cotton swabs in time. (5) Seedling hardening and germplasm screening: The grafted seedlings were transferred to the hardening medium (MS basal medium + 20g / L sucrose + 7g / L agar, pH 5.8) for 6 days of hardening. The hardening temperature was 23℃, the humidity was 75% and natural diffused light was used. Then the grafted seedlings were transplanted into sterile substrate soil (peat soil: perlite = 3:1) and managed in the greenhouse (temperature 23℃, humidity maintained at 65%) until the potato tubers matured. Potato seed tubers were harvested to obtain the newly created potato germplasm.

[0035] Comparative Example 1 differs from Example 1 in that: in step (1), a potato variety with common traits is selected as the rootstock, while other conditions remain unchanged.

[0036] The difference between Comparative Example 2 and Example 1 is that in step (1), the parent tissue culture seedlings are cultured at room temperature and under natural light, while other conditions remain unchanged.

[0037] The difference between Comparative Example 3 and Example 1 is that in step (2), no seedling hardening pretreatment was performed 3 days before grafting, while other conditions remained unchanged.

[0038] The difference between Comparative Example 4 and Example 1 is that in step (2), 10 mg / L 6-benzylaminopurine (6-BA) was not added to the grafting pretreatment solution, while other conditions remained unchanged.

[0039] The difference between Comparative Example 5 and Example 1 is that in step (2), the grafting pretreatment solution is not applied lightly to the cut of the rootstock seedling and the base of the scion, while other conditions remain unchanged.

[0040] The difference between Comparative Example 6 and Example 1 is that in step (3), the biodegradable glutinous rice paper grafting film is not tightly wrapped, while other conditions remain unchanged.

[0041] The difference between Comparative Example 7 and Example 1 is that in step (4), 10 mg / L NAA was not added to the callus culture medium, while other conditions remained unchanged.

[0042] The difference between Comparative Example 8 and Example 1 is that in step (4), from day 0 to day 20, the conditions for the entire process of grafted seedling callus culture are 25±1℃ and in the dark, while other conditions remain unchanged.

[0043] The difference between Comparative Example 9 and Example 1 is that in step (4), the conditions for the entire process of grafted seedling callus culture from day 0 to day 20 are: 23±2℃, light intensity 2000 lux, light duration 14h / d, and other conditions remain unchanged.

[0044] The difference between Comparative Example 10 and Example 1 is that in step (5), the seedling temperature is 30°C, the humidity is 88%, and the light is natural, while other conditions remain unchanged.

[0045] Comparative Example 11 differs from Example 1 in that: in step (5), the seedling hardening medium is MS basal medium + 30 g / L sucrose + 5 g / L agar, pH is 7.0, and other conditions remain unchanged.

[0046] The difference between Comparative Example 12 and Example 1 is that in step (5), the sterile substrate soil is peat soil: perlite = 1:1, and other conditions remain unchanged.

[0047] The difference between Comparative Example 13 and Example 1 is that in step (2), the tissue culture medium contains only MS medium, while other conditions remain unchanged.

[0048] The efficacy test adopted the creation methods of Examples 1-3 and Comparative Examples 1-12, respectively. 150 plants were grafted on 18 adjacent planting plots, with 3 replicates in each group, to create germplasm. The soil properties, breeding management methods and breeding environment of the 18 adjacent planting plots were the same. The yield of robust tissue culture seedlings (%), grafting survival rate (%), hardening-off survival rate (%), and seed potato harvest (kg / mu) were recorded.

[0049] The yield of robust tissue culture seedlings (%) = number of robust tissue culture seedlings / (number of rootstocks + number of scions) × 100%; the grafting survival rate (%) = number of grafted seedlings that survive / number of grafts × 100%; the hardening-off survival rate (%) = number of hardened-off seedlings that survive / number of hardened-off seedlings × 100%. The experimental results are shown in Table 1 below.

[0050] Table 1 As shown in Table 1, Comparative Example 1 demonstrates, selecting rootstocks with specific traits (such as high resistance) is beneficial for obtaining new germplasm with the desired characteristics of high yield and high resistance; Comparative Example 2 illustrates that the culture method affects the quality of tissue culture seedlings and is related to subsequent grafting and hardening-off survival rates; Comparative Examples 3-5 This indicates that the grafting treatment method directly affects the grafting survival rate. The absence of hormones (6-BA) and pretreatment solutions (Comparative Examples 4 and 5) inhibits callus formation and vascular bundle connection. In Comparative Example 6 (inadequate bandaging), the graft union failed to adhere tightly, leading to dehydration or contamination and a lower survival rate. In Comparative Example 7, the lack of auxin severely inhibited callus growth, resulting in poor graft union healing and a low survival rate. Darkness (Comparative Example 8) or continuous light (Comparative Example 9) are not favorable conditions for callus formation, while the phased light-based culture strategy of the present invention (dark culture followed by light culture) promotes callus and subsequent bud development. Inappropriate temperature and humidity (Comparative Example 10), unsuitable hardening-off culture medium (Comparative Example 11), or changes in transplanting substrate (Comparative Example 12) all increase environmental stress to varying degrees, reduce survival rate, and ultimately affect yield. Comparative Example 13, compared to Example 1, shows that the tissue culture medium of the present invention can induce beneficial variations in rootstock tissues and enhance the physiological activity and final economic traits of grafted seedlings.

[0051] In summary, this invention shortens the stabilization cycle of new germplasm traits by optimizing key steps such as tissue culture seedling cultivation, grafting operations, callus culture, and hardening-off, without seasonal limitations, thus doubling breeding efficiency and achieving efficient creation of new germplasm. By purposefully selecting tissue culture seedlings with specific traits (such as high resistance and excellent quality) as rootstocks or scions, the trait variation of newly created germplasm becomes more directional and predictable, effectively avoiding the generation of a large number of invalid variations in mutation breeding, thereby significantly reducing the scale and cost of later screening. Grafting can quickly express new traits generated by the interaction between rootstock and scion in the grafted organism, providing an efficient technical means for rapidly obtaining potatoes with specific traits. Asexual grafting can bypass sexual reproduction barriers such as self-incompatibility and inbreeding depression in potatoes, enabling the fusion of traits between parental materials that are difficult to exchange genes through hybridization. This opens up new avenues for utilizing superior genes from special germplasm resources such as wild species and local varieties. By utilizing the low-frequency variations that may spontaneously arise in plant somatic cells during tissue culture and grafting healing, additional genetic diversity can be added to the creation of new germplasm. It may be possible to obtain new traits that are difficult to achieve through sexual hybridization, thus broadening the genetic basis of potato breeding.

[0052] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for creating new germplasm by grafting potato tissue culture seedlings, characterized in that, Includes the following steps: (1) Parental tissue culture seedling cultivation: Select potato varieties with the target traits as rootstocks and potato varieties with ordinary traits as scions. Take disease-free stem tips as explants. After disinfecting the explants, inoculate them into MS basal medium and culture them to obtain scion seedlings and rootstock seedlings respectively; (2) Pre-grafting treatment: Harden off the scion seedlings and rootstock seedlings. Cut off the terminal bud of the rootstock seedlings, cut the rootstock seedlings into stem segments, remove the leaves, lightly apply grafting pretreatment solution to the cut of the rootstock seedlings and the base of the scion, let it stand, and then insert the end near the root into the tissue culture seedling. (2) Cultivation in a culture medium to obtain pretreated scion seedlings and rootstock seedlings; (3) Grafting of tissue culture seedlings: graft the base of the scion into the cut of the rootstock, and tightly wrap the graft union with biodegradable glutinous rice paper grafting film; (4) Callus culture of grafted seedlings: inoculate the grafted seedlings into the callus culture medium for grafted seedling callus culture; (5) Hardening and acclimatization and germplasm screening: transfer the grafted seedlings to the hardening culture medium for hardening, and then transplant the grafted seedlings into sterile substrate soil, and manage them in the greenhouse until the potato tubers mature, harvest the potato seed tubers, and obtain the newly created potato germplasm.

2. The method according to claim 1, characterized in that, In step (1), the explant is 2-3 cm long and has 1-2 axillary buds; the target trait is one or more of the following: resistance, high starch content, and high protein content.

3. The method according to claim 1, characterized in that, In step (1), the disinfection is first soaked in 75% ethanol for 30-45 seconds, then disinfected with 0.1% mercuric chloride solution for 5-6 minutes, and then rinsed with sterile water 4-5 times.

4. The method according to claim 1, characterized in that, In step (1), the MS basal medium is supplemented with 30 g / L sucrose and 7 g / L agar, and the pH is 5.9; the culture is carried out in a culture room with conditions of 20±1℃, light intensity of 2000~2500 lux, and light duration of 14h / d for 20~30 days; the scion seedlings and rootstock seedlings are robust tissue culture seedlings with a plant height of 8~10cm and a stem diameter of 0.1~0.2cm.

5. The method according to claim 1, characterized in that, In step (2), the seedling hardening pretreatment involves transferring the scion seedlings and rootstock seedlings to a cultivation environment with a light intensity of 3000 lux 3 days before grafting; the rootstock seedlings are cut into stem segments of 2.8 to 3.2 cm in length before grafting.

6. The method according to claim 1, characterized in that, In step (2), the grafting pretreatment solution is prepared by dissolving 20 mg / L naphthaleneacetic acid and 10 mg / L 6-benzylaminopurine in sterile water and stirring until homogeneous. The standing time is 10-15 min. The tissue culture medium is prepared from the following raw materials in parts by weight: 4.2-4.7 parts MS medium, 28-33 parts sucrose, 0.05-0.15 parts inositol, 6-8 parts agar, 0.1-0.2 parts vitamin B1, 0.1-0.3 parts vitamin C, 0.1-0.2 parts vitamin B6, 0.8-1.3 parts 0.5-2 mg / L 6-benzylaminopurine, and 0.05-0.2 parts 0.1-0.5 mg / L naphthaleneacetic acid. The materials are mixed evenly, the pH is adjusted to 5.6-5.8, and sterilized.

7. The method according to claim 1, characterized in that, In step (3), the grafting adopts the "cleft grafting method". The specific steps are as follows: on a sterile operating table, use a sterile scalpel to cleft the top of the rootstock stem segment downward along the central axis. The cut depth is 1.5-2cm and the cut width is 0.1-0.2cm. The cut width matches the diameter of the scion base. Cut the base of the scion into a wedge shape with a length of 1.5-2cm. Insert the base of the scion into the cut of the rootstock to complete the grafting. The wrapping is wrapped 2-3 times.

8. The method according to claim 1, characterized in that, In step (4), the callus culture medium is MS basal medium + 15 g / L sucrose + 7 g / L agar + 10 mg / L NAA + 5 mg / L 6-BA, with a pH of 5.

8.

9. The method according to claim 1, characterized in that, In step (4), the grafted seedling callus culture is as follows: in the early stage of callus culture, the culture conditions are 25±1℃ and dark environment for 0 to 7 days; after 7 days, the conditions are adjusted to 23±2℃, light intensity of 2000 lux and light time of 14h / d.

10. The method according to claim 1, characterized in that, In step (5), the seedling hardening culture medium is MS basal medium + 20 g / L sucrose + 7 g / L agar, with a pH of 5.8; the hardening time is 5 to 7 days, and the conditions are: temperature 20 to 25℃, humidity 70% to 80%, and natural diffused light; the sterile substrate soil is peat soil and perlite in a mass ratio of 3:1; the temperature of the greenhouse under normal management is 22 to 25℃, and the humidity is maintained at 60% to 70%.

Images