Method and device for creating new potato germplasm with high resistance to late blight by inter-species grafting

Abstract

The application discloses a method and device for creating high-late-blight-resistant potato new germplasm through inter-specific grafting, which comprises the following steps: (1) cultivating potato seedlings and late-blight-resistant tomato seedlings; (2) grafting each other as a stock and a scion; (3) treating the grafted plant to obtain grafted seedlings 1; (4) screening high-late-blight-resistant germplasm: S1, inoculating a small amount of pythium on the grafted seedlings 1 after sodium chloride stress, screening out plants with certain disease resistance, moving the plants to outdoor culture, and timely harvesting tubers to obtain tubers 1; S2, grafting the tubers 1 with tomatoes after planting, obtaining grafted seedlings 2, inoculating a large amount of pythium on the grafted seedlings 2, and performing high-temperature and high-humidity treatment, screening out plants with strong disease resistance, moving the plants to outdoor culture, and timely harvesting tubers to obtain tubers 2; S3, repeating steps 1-2 of S2 on the tubers 2 for 1-2 times, and harvesting the tubers, which are the required high-late-blight-resistant potato new germplasm. The application obtains high-late-blight-resistant and genetically stable potato new germplasm, shortens the variety breeding time and reduces the cost.

Description

Technical Field

[0001] This invention belongs to the field of breeding technology, and specifically relates to a method and apparatus for creating new potato germplasm with high resistance to late blight through interspecific grafting. Background Technology

[0002] Late blight of potatoes is a severe and widespread disease that can lead to significant yield reductions or even total crop failure. In China, late blight causes approximately 10-15% yield reductions annually, with severely affected fields experiencing complete crop failure and resulting in substantial economic losses. Late blight primarily occurs under hot and humid conditions. In winter potato-growing areas of my country, it mainly occurs during the rainy season from February to April when temperatures rise, while in single- and double-cropping potato areas, it occurs after rains from May to September. Late blight primarily affects potato petioles, stems, and tubers, but leaves are most susceptible. When leaves are infected, small dark green spots first appear at the leaf tips or margins. Under humid conditions, these spots rapidly expand, becoming irregular, water-soaked, dark green. Once the petioles and stems are also infected, the leaves wither and droop, eventually leading to the entire plant turning black and rotting until the stem collapses and the plant dies.

[0003] Compared to chemical control and field management, the breeding and application of disease-resistant varieties is the most effective and safest way to prevent potato late blight. Currently, late blight-resistant varieties are mainly developed through hybridization breeding, selection breeding, mutation breeding, and genetic engineering. However, all of these methods are time-consuming and difficult to meet production demands. Grafting involves joining the incisions of two plants together to allow them to heal and form a new composite plant. In agriculture, grafting is mainly used to increase yield and stress resistance. Many studies have shown that after grafting, there is exchange of substances within the plant. Plant hormones, small molecule metabolites, inorganic salts, sugars, proteins, DNA, and RNA can all be transferred between the rootstock and scion. Therefore, grafting can alter the plant's gene expression. Some patents disclose the use of grafting technology to control potato late blight, such as patent "A method for controlling potato late blight using grafting technology" (CN 109156188 B). This method uses a potato variety highly resistant to late blight as the scion and a potato variety with good marketability and high market demand but low resistance to late blight as the rootstock. After grafting, no chemical agents need to be applied throughout the potato's growth period, achieving biological control of potato late blight and realizing green and pollution-free production. However, this grafting method can only be applied in the current production season and cannot obtain heritable potato germplasm resistant to late blight for long-term production. It cannot solve the problems of the urgent need for stable late blight-resistant varieties in potato production and the long and costly process of variety breeding.

[0004] On the other hand, existing potato aeroponic systems typically place a 2-3cm thick foam board on top of the aeroponic box, with planting holes in the board for planting the seedlings. After grafting potatoes and tomatoes, the grafting site is usually secured with clips, which are then placed over the planting holes to keep the grafting site above the foam board, preventing it from falling into the box and rotting due to nutrient solution contamination. However, in the early stages of grafting, the grafting site is still unhealed and has wounds, making it highly susceptible to contamination and rotting from the nutrient solution sprayed directly from the bottom of the box through the planting holes, thus reducing the survival rate of the grafted seedlings. Summary of the Invention

[0005] The purpose of this invention is to provide a method and apparatus for creating new potato germplasm highly resistant to late blight through interspecific grafting, solving the problems of the lack of current technology for creating potato germplasm resistant to late blight using grafting and the excessively long and costly time required for conventional variety breeding. The specific technical solution is as follows:

[0006] A method for creating new potato germplasm highly resistant to late blight through interspecific grafting includes the following steps:

[0007] (1) Cultivation of potato seedlings and tomato seedlings:

[0008] After the seeds of late blight-resistant tomatoes are pre-germinated, they are sown in seedling trays containing seedling substrate. When the seeds have grown to two compound leaves, they are transferred to a self-made aeroponic device and continued to grow until they have three to four compound leaves. Conventional aeroponic management is then used to cultivate the plants.

[0009] Select virus-free potato seed tubers that have already sprouted, have good market demand, but are not resistant to or have low resistance to late blight, and plant them in flowerpots with seedling substrate. Under natural light, cultivate them until they grow to 4-5 compound leaves. Then cut them at the joint of the seed tuber, keeping 2-3 compound leaves, soak them in rooting solution for 25-35 minutes, and transplant them into a self-made aeroponic device. Before they have rooted, only spray them with rooting solution containing rooting powder. When the roots are 1-2 cm long, start spraying nutrient solution and carry out conventional aeroponic management to cultivate the plants.

[0010] (2) Grafting with each other as rootstock and scion: Use potatoes with good marketability and high market demand but not resistant or low resistant to late blight as rootstock and tomatoes resistant to late blight as scions, or use tomatoes resistant to late blight as rootstock and potatoes with good marketability and high market demand but not resistant or low resistant to late blight as scions.

[0011] The grafting operation includes: the method is cleft grafting, which is to cut the rootstock stem at a thick part under shaded conditions and split it in the middle with a 1-2cm opening. Then, cut off the lower end of the scion of similar thickness, retaining 1-2 functional leaves. Cut the scion stem segment into a gentle slope, keeping both sides symmetrical and the length equal to the opening of the rootstock. Then insert the scion into the rootstock so that the cut surface of the rootstock and the cut surface of the scion are in close contact, and fix it with a grafting clip.

[0012] (3) Treatment of grafted plants: Grafted seedlings of late blight resistant tomatoes / non-resistant or low-resistant potatoes (scions / rootstocks), or non-resistant or low-resistant potatoes / resistant tomatoes, are further cultivated in aerosol growing devices. The ambient temperature of the aerosol growing device is controlled at 20-24℃ and the relative humidity at 60-70% to prevent the grafted seedlings from dehydrating and dying. In the first week, the seedlings are covered with a shade net with a shading rate of 70-80%, and then exposed to natural light for 3-4 weeks. During this period, all the runners are cut off to obtain 1 grafted seedling.

[0013] (4) Screening of new germplasm with high resistance to late blight:

[0014] S1. First, the grafted seedling 1 is subjected to sodium chloride stress, then a small amount of Phytophthora inoculated, and plants with certain disease resistance are selected and moved outdoors for cultivation. The tubers are harvested in time to obtain tuber 1.

[0015] S2. After cultivating the tuber 1 and the late blight resistant tomato according to step (1) and grafting them as rootstock and scion according to step (2), the grafted seedling 2 is obtained by managing them according to step (3). After inoculating the grafted seedling 2 with a large amount of Phytophthora, the seedling is subjected to high temperature and high humidity treatment. Plants with high resistance to late blight are selected and moved outdoors for cultivation. The tubers are harvested in time to obtain tuber 2.

[0016] The high temperature and high humidity treatment is as follows: first, adjust the temperature to 26-30℃ and incubate under light for 14-16 hours, then adjust the temperature to 18-22℃ and incubate in the dark for 8-10 hours. The humidity is maintained at 90-95% throughout the process, and this cycle is repeated for 7-10 days.

[0017] S3. Repeat step S2 1-2 times with the tuber 2 to harvest the tuber, which is the desired new potato germplasm with high resistance to late blight.

[0018] Further, in step (1), the aeroponic device includes: a first aeroponic box, a second aeroponic box, a first spray system, a second spray system, a planting plate, and an annular rubber pad;

[0019] The first aeroponic box is positioned above the second aeroponic box, and the first and second aeroponic boxes are connected by the planting plate. The top of the first aeroponic box is open. The first spray system is located inside the first aeroponic box, and the second spray system is located inside the second aeroponic box. Planting holes are spaced apart on the planting plate, and an annular rubber gasket is positioned above the planting plate, corresponding vertically to the planting holes. The annular rubber gasket includes a first gasket and a second gasket stacked together, with the first gasket positioned above the second gasket. The inner diameter of the first gasket is smaller than the inner diameter of the second gasket, and the inner circles of the first and second gaskets are provided with easy-tear openings.

[0020] Furthermore, the annular rubber gasket also includes a third gasket, which is stacked between the first gasket and the second gasket. The inner diameter of the third gasket is larger than the inner diameter of the first gasket and smaller than the inner diameter of the second gasket. The inner circle of the third gasket is also provided with an easy-tear opening.

[0021] Furthermore, the nozzle of the second spray system extends downwards by 10-20 cm from the junction of the planting plate and the second aeroponic box at a 30-45 degree angle to spray.

[0022] Furthermore, in step (2), the grafting time is selected within one week before or after New Year's Day.

[0023] Further, in step (4) S1, the sodium chloride stress is to spray one leaf of the grafted seedling 1-2 times a day for 2-3 consecutive days with a sodium chloride solution of 100-150 mmol / L, each time until moist; the inoculation of a small amount of Phytophthora refers to using a spore concentration of 2×10 4 Spray one leaf of grafted seedlings with a Phytophthora suspension of 1-2 mL per plant.

[0024] Further, in step (4) S1, the plant with certain disease resistance refers to a plant with one of the following diseases: the disease is that the whole plant leaves have no disease spots, individual leaves have individual disease spots, and less than 1 / 4 of the whole plant leaves have disease spots.

[0025] Further, in step (4) S2, the method for inoculating the large quantity of Phytophthora is: using a spore concentration of 2×10⁻⁶. 4 Spray two leaves of grafted seedlings with a Phytophthora suspension of 5-10 mL per plant.

[0026] Furthermore, in step (4) S2, the plant with high resistance to late blight refers to a plant with no disease spots on all leaves or with individual disease spots on individual leaves.

[0027] Furthermore, the aeroponic device is placed in a temperature-controlled air-conditioned room for temperature and humidity control treatment, and / or, a temperature control device, a light control device, and a humidity sensor are provided in the first aeroponic chamber. The temperature control device includes a temperature probe, a heating element, and a temperature controller. The temperature probe and the heating element are electrically connected to the temperature controller, which controls the temperature inside the first aeroponic chamber. The light control device includes an LED light and a timer. The LED light and the timer are connected, and the timer controls the illumination time inside the first aeroponic chamber. The humidity sensor is connected to the first spray system to control the air humidity inside the first aeroponic chamber.

[0028] Compared with existing technologies, the present invention has the following advantages:

[0029] 1. This invention involves grafting late blight-resistant tomato seedlings with 3-4 compound leaves with late blight-resistant potato seedlings with 4-5 compound leaves. These seedlings are of good market quality and in high demand, but are not resistant or have low resistance to late blight. After grafting, a self-made aeroponic device is used for aeroponic management, and late blight-resistant germplasm is screened to breed the desired new potato germplasm with high resistance to late blight. This solves the problems of the current lack of technology for creating new potato germplasm resistant to late blight using grafting methods, as well as the long time and high cost of conventional variety breeding.

[0030] 2. The aeroponic device of the present invention, by providing annular rubber pads on the planting board, can reduce the contact between the grafting wound and the water vapor coming out of the box, reduce disease and rot, and improve the survival rate of grafted seedlings; the nutrient solution nozzle extends downward at an angle of 30-45 degrees for about 15 cm from the bottom of the planting board and the junction of the box, on the one hand, avoiding the nozzle from spraying directly into the planting holes on the planting board and causing contamination of the grafting interface, and on the other hand, it can spray at close range, spraying more evenly on the roots, allowing the roots to absorb nutrients more efficiently, promoting faster healing of the grafting interface and plant growth.

[0031] 3. In screening new potato germplasm with high resistance to late blight, this invention first subjectes grafted seedling 1 to sodium chloride stress, which increases the expression of relevant resistance genes in seedling 1. Then, a small amount of Phytophthora inoculation is performed. Under the stimulation of a small amount of Phytophthora, the self-defense of grafted seedling 1 is stronger, and the expression of relevant resistance genes is greater, accelerating the transmission of relevant genes between rootstock and scion. This allows for the preliminary screening of plants with certain disease resistance. Grafted seedling 2 is then inoculated with a large amount of Phytophthora and subjected to high temperature and humidity treatment to create an environment conducive to late blight outbreaks, further promoting the expression and transmission of disease resistance genes. Simultaneously, multiple generations of continuous grafting and screening of harvested potato materials ensure that genes from late blight-resistant tomatoes are more fully expressed, rapidly transmitted, and stabilized. Ultimately, a new potato germplasm with stable disease resistance traits and high resistance to late blight is selected, which is more beneficial for production and application. Attached Figure Description

[0032] 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. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0033] Figure 1 This is a schematic diagram of the aeroponic device of the present invention.

[0034] Figure 2 This is a schematic diagram of the internal structure of the aeroponic device of the present invention.

[0035] Figure 3 This is a schematic diagram of the structure of the annular rubber gasket of the present invention.

[0036] Figure 4 This is a schematic diagram showing the disassembled structure of the annular rubber gasket of the present invention.

[0037] Figure 5 The new germplasm material created in Example 2 showed a late blight disease severity level of 0 in the field.

[0038] Figure 6 The new germplasm material created in Example 3 showed a late blight disease severity level of 1 in the field.

[0039] Figure 7 The new germplasm material created in Comparative Example 1 showed a late blight disease severity level of 3 in the field.

[0040] The main reference numerals in the attached figures are explained as follows:

[0041] 1. First aeroponic box; 2. Second aeroponic box; 3. Planting board; 31. Planting hole; 4. Annular rubber gasket; 41. First gasket; 42. Second gasket; 43. Third gasket; 44. Easy-tear opening; 5. Temperature control device; 51. Temperature probe; 52. Heating element; 53. Temperature controller; 6. Light control device; 61. LED light; 62. Time controller; 7. First spray system; 8. Second spray system; 81. Nozzle; 9. Blackout curtain; 10. Collection door; 11. Humidity sensor. Detailed Implementation

[0042] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0043] In the description of this invention, it should be noted that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "top surface," "bottom surface," "inner," "outer," "inner side," and "outer side," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0044] In the description of this invention, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number. Where the terms "first," "second," and "third" are used for descriptive purposes and to distinguish technical features, they should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the sequential relationship of the indicated technical features.

[0045] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances. The following describes embodiments of the invention based on its overall structure.

[0046] Example 1

[0047] Combination Figure 1-2 An aeroponic device includes: a first aeroponic box, a second aeroponic box, a first spray system, a second spray system, a planting plate, and an annular rubber pad; the first aeroponic box is disposed above the second aeroponic box, and the first aeroponic box and the second aeroponic box are connected by the planting plate;

[0048] The first aeroponics chamber is equipped with a first spray system, a temperature control device, a light control device, and a humidity sensor. The first spray system is used to control the air humidity inside the first aeroponics chamber. The temperature control device is used to control the temperature inside the first aeroponics chamber. The light control device is used to control the light inside the first aeroponics chamber. The humidity sensor is used to sense the humidity inside the first aeroponics chamber. The top of the first aeroponics chamber is set with an opening, which can be used to spray sodium chloride solution or Phytophthora suspension.

[0049] The second aeroponic box is equipped with a second spray system, which is located at all four corners. This system is used to periodically spray water or nutrient solution onto the roots and tubers of the plants inside the second aeroponic box. The second aeroponic box is also equipped with a light-blocking curtain, a collection door, and other structures.

[0050] The planting board is provided with planting holes at intervals. The planting board is used to plant crops. The stems and leaves of the crops grow in the first aeroponic box above the planting board, and the roots and tubers of the crops grow in the second aeroponic box below the planting board.

[0051] The annular rubber pad is positioned above the planting board and corresponds vertically to the position of the planting hole; the annular rubber pad is used to block water vapor from the second aeroponic box and to fix the crops on the planting board.

[0052] Specifically, in combination Figure 3-4 The annular rubber gasket comprises a first gasket, a second gasket, and a third gasket stacked together. The first gasket is positioned above the second gasket, and its inner diameter is smaller than that of the second gasket. Both the first and second gaskets have tear-off openings on their inner circumferences. The third gasket is stacked between the first and second gaskets, and its inner diameter is larger than that of the first gasket but smaller than that of the second gasket. The third gasket also has tear-off openings on its inner circumference. The annular rubber gasket is made of rubber and has a certain degree of elasticity, allowing it to adapt to crop growth. The tear-off openings allow the gasket to be removed according to the growth of the crop stem. Preferably, the tear-off openings of the first, second, and third gaskets are staggered.

[0053] Combination Figure 2 The nozzles of the second spray system extend downwards at a 30-45 degree angle, 10-20 cm from the junction of the planting board and the second aeroponic box, to spray. This avoids direct spraying from below, which could easily enter the planting holes on the planting board and contaminate the grafting interface. Furthermore, it allows for close-range spraying, ensuring more even distribution of nutrients to the roots, enabling more efficient nutrient absorption and promoting faster healing of the grafting interface and plant growth.

[0054] Furthermore, the temperature control device includes a temperature probe, a heating tube, and a temperature controller. The temperature probe and the heating tube are electrically connected to the temperature controller, which controls the temperature inside the first aeroponic chamber.

[0055] Furthermore, the light control device includes an LED lamp and a time controller, which are electrically connected to each other, and the time controller controls the illumination time in the first aeroponic chamber.

[0056] Furthermore, the humidity sensor is connected to the first spray system to control the air humidity inside the first aeroponic chamber.

[0057] In another embodiment, the first aeroponic box is also replaced by a support frame. When the support frame is used, the temperature, light and humidity of the plant stems and leaves are controlled by equipment in the air-conditioned room.

[0058] This invention relates to an aeroponic device for cultivating grafted potato-tomato seedlings. By incorporating a ring-shaped rubber pad on the planting board, it blocks moisture from the second aeroponic chamber and elevates the grafting wound, reducing contact between the grafting wound and moisture emanating from the chamber. This reduces disease and rot, improving the survival rate of the grafted seedlings. The nutrient solution spray nozzle extends downwards at a 30-45 degree angle, approximately 15 cm, from the junction of the planting board and the chamber. This design avoids direct spraying from below, which could easily enter the planting holes on the planting board and contaminate the grafting interface. Furthermore, it allows for close-range spraying, ensuring more even distribution of nutrients to the roots, enabling more efficient nutrient absorption and promoting faster healing of the grafting interface and overall plant growth.

[0059] Example 2

[0060] Material cultivation and grafting were carried out in the potato aeroponic chamber of the Institute of Economic Crops, Guangxi Academy of Agricultural Sciences, under natural light, at a temperature of 20℃ and a relative humidity of 60%. The potato aeroponic chamber was equipped with multiple aeroponic devices as described in Example 1. The potato material was "Feiwuruita," a variety with good marketability but not resistant to late blight, while the tomato material resistant to late blight was 2A37. "Feiwuruita" could be purchased from the Guangxi Academy of Agricultural Sciences or the general market, while 2A37 tomato was a variety bred by Guangxi University. Phytophthora was obtained from the Institute of Microbiology, Guangxi Academy of Agricultural Sciences.

[0061] A method for creating new potato germplasm highly resistant to late blight through interspecific grafting includes the following steps:

[0062] (1) Cultivation of potato seedlings and tomato seedlings:

[0063] After the seeds of 2A37 tomatoes were pre-germinated, they were sown in seedling trays containing seedling substrate. When the seeds grew to 2 compound leaves, they were transferred to a self-made aeroponic device and continued to grow until they had 3-4 compound leaves. Conventional aeroponic management was then used to cultivate the plants.

[0064] Select virus-free "Feiwurui" potato seed tubers that have already sprouted and plant them in flowerpots with seedling substrate. Under natural light, cultivate them until they grow to 4-5 compound leaves. Then cut them at the connection point of the seed tubers, keeping 2-3 compound leaves. Soak them in rooting solution for 25 minutes and transplant them into a self-made aeroponic device. Before they have rooted, only spray them with rooting solution containing rooting powder. When the roots are 1-2 cm long, start spraying nutrient solution and carry out conventional aeroponic management to cultivate the plants.

[0065] (2) Grafting with each other as rootstock and scion: Use 2A37 tomato as rootstock and "Feiwuruita" as scion; the grafting time is within one week before New Year's Day;

[0066] The grafting operation includes: the method is cleft grafting, which is to cut the rootstock stem at a thick part under shaded conditions and split it in the middle with a 1-2cm opening. Then, cut off the lower end of the scion of similar thickness, retaining 1-2 functional leaves. Cut the scion stem segment into a gentle slope, keeping both sides symmetrical and the length equal to the opening of the rootstock. Then insert the scion into the rootstock so that the cut surface of the rootstock and the cut surface of the scion are in close contact, and fix it with a grafting clip.

[0067] (3) Treatment of grafted plants: The grafted “Feiwuruita” / 2A37 tomato (scion / rootstock) seedlings were further cultivated in an aeroponic device. The ambient temperature of the aeroponic device was controlled at 20℃ and the relative humidity at 60% to prevent the grafted seedlings from dehydrating and dying. During the first week, the seedlings were covered with a shade net with a 70% shading rate, and then naturally exposed to light for another 4 weeks. During this period, all the runners were cut off to obtain 1 grafted seedling.

[0068] (4) Screening of new germplasm with high resistance to late blight:

[0069] S1. First, the grafted seedling 1 is subjected to sodium chloride stress, then a small amount of Phytophthora inoculation is performed to screen out plants with certain disease resistance. These plants are then moved outdoors for cultivation, and the tubers are harvested in a timely manner to obtain tuber 1. The plants with certain disease resistance refer to plants with one of the following diseases: no disease spots on all leaves, individual disease spots on a few leaves, or disease spots on less than 1 / 4 of the leaves of the whole plant.

[0070] The sodium chloride stress was achieved by spraying one leaf of the grafted seedling once daily for two consecutive days with a 100 mmol / L sodium chloride solution, spraying until the leaves were moist each time; the inoculation with a small amount of Phytophthora refers to using a spore concentration of 2 × 10⁻⁶. 4 Spray one leaf of each grafted seedling with a Phytophthora suspension of 1 / mL, with a spraying amount of 1mL per plant.

[0071] S2. After cultivating the tuber 1 and 2A37 tomato according to step (1) and grafting them as rootstock and scion according to step (2), the grafted seedling 2 is obtained by managing them according to step (3). After inoculating the grafted seedling 2 with a large amount of Phytophthora, the plant is subjected to high temperature and high humidity treatment. Plants with no disease spots on all leaves or with individual disease spots on individual leaves are selected and moved outdoors for cultivation. The tubers are harvested in time to obtain tuber 2.

[0072] The high temperature and high humidity treatment is as follows: first, the temperature of the first aeroponic chamber is adjusted to 26℃ and cultured under light for 14 hours, then the temperature is adjusted to 18℃ and cultured in the dark for 10 hours. Throughout the process, the humidity of the first aeroponic chamber is maintained at 90%, and this cycle is repeated for 7 days.

[0073] The inoculation method for the large quantity of Phytophthora is as follows: using spores at a concentration of 2 × 10⁻⁶. 4 Spray two leaves of grafted seedlings with a Phytophthora suspension of 5 mL / mL, with a spraying amount of 5 mL per seedling.

[0074] S3. Repeat step S2 once with the tuber 2 and harvest the tuber, which is the desired new potato germplasm with high resistance to late blight.

[0075] Example 3

[0076] Material cultivation and grafting were carried out in the potato aeroponic chamber of the Institute of Economic Crops, Guangxi Academy of Agricultural Sciences, under natural light, at a temperature of 24℃ and a relative humidity of 70%. The potato aeroponic chamber was equipped with multiple aeroponic devices as described in Example 1. The potato material was 'Feiwuruita,' a variety with good marketability but not resistant to late blight, while the tomato material resistant to late blight was 2A37. 'Feiwuruita' was available from the Guangxi Academy of Agricultural Sciences or in general markets, while 2A37 tomato was a variety bred by Guangxi University. Phytophthora was obtained from the Institute of Microbiology, Guangxi Academy of Agricultural Sciences.

[0077] A method for creating new potato germplasm highly resistant to late blight through interspecific grafting includes the following steps:

[0078] (1) Cultivation of potato seedlings and tomato seedlings:

[0079] After the seeds of 2A37 tomatoes were pre-germinated, they were sown in seedling trays containing seedling substrate. When the seeds grew to 2 compound leaves, they were transferred to a self-made aeroponic device and continued to grow until they had 3-4 compound leaves. Conventional aeroponic management was then used to cultivate the plants.

[0080] Select virus-free "Feiwuruita" seed potatoes that have already sprouted and plant them in flowerpots with seedling substrate. Under natural light, cultivate them until they grow to 4-5 compound leaves. Then cut them at the connection point of the seed potatoes, keeping 2-3 compound leaves. Soak them in rooting solution for 25-35 minutes and transplant them into a self-made aeroponic device. Before they have rooted, only spray them with rooting solution containing rooting powder. When the roots are 1-2 cm long, start spraying nutrient solution and carry out conventional aeroponic management to cultivate the plants.

[0081] (2) Grafting with each other as rootstock and scion: Use "Feiwurui" as rootstock and 2A37 tomato as scion; the grafting time is within one week after New Year's Day;

[0082] The grafting operation includes: the method is cleft grafting, which is to cut the rootstock stem at a thick part under shaded conditions and split it in the middle with a 1-2cm opening. Then, cut off the lower end of the scion of similar thickness, retaining 1-2 functional leaves. Cut the scion stem segment into a gentle slope, keeping both sides symmetrical and the length equal to the opening of the rootstock. Then insert the scion into the rootstock so that the cut surface of the rootstock and the cut surface of the scion are in close contact, and fix it with a grafting clip.

[0083] (3) Treatment of grafted plants: The grafted 2A37 tomato / "Feiwuruita" (scion / rootstock) seedlings were further cultivated in an aeroponic device. The ambient temperature of the aeroponic device was controlled at 24℃ and the relative humidity at 70% to prevent the grafted seedlings from dehydrating and dying. During the first week, the seedlings were covered with a shade net with a shading rate of 80%, and then naturally exposed to light for another 3 weeks. During this period, all the runners were cut off to obtain 1 grafted seedling.

[0084] (4) Screening of new germplasm with high resistance to late blight:

[0085] S1. First, the grafted seedling 1 is subjected to sodium chloride stress, then a small amount of Phytophthora inoculation is performed to screen out plants with certain disease resistance. These plants are then moved outdoors for cultivation, and the tubers are harvested in a timely manner to obtain tuber 1. The plants with certain disease resistance refer to plants with one of the following diseases: no disease spots on all leaves, individual disease spots on a few leaves, or disease spots on less than 1 / 4 of the leaves of the whole plant.

[0086] The sodium chloride stress was achieved by spraying one leaf of the grafted seedling twice daily for three consecutive days with a 150 mmol / L sodium chloride solution, each time spraying until the leaf was moist. The inoculation with a small amount of Phytophthora refers to using a spore concentration of 2 × 10⁻⁶. 4 Spray one leaf of the grafted seedling with a Phytophthora suspension of 1 / mL, with a spraying amount of 2mL per plant.

[0087] S2. After cultivating the tuber 1 and 2A37 tomato according to step (1) and grafting them as rootstock and scion according to step (2), the grafted seedling 2 is obtained by managing them according to step (3). After inoculating the grafted seedling 2 with a large amount of Phytophthora, the plant is subjected to high temperature and high humidity treatment. Plants with no disease spots on all leaves or with individual disease spots on individual leaves are selected and moved outdoors for cultivation. The tubers are harvested in time to obtain tuber 2.

[0088] The high temperature and high humidity treatment is as follows: first, the temperature of the first aeroponic chamber is adjusted to 30℃ and cultured under light for 16 hours, then the temperature is adjusted to 22℃ and cultured in the dark for 8 hours. Throughout the process, the humidity of the first aeroponic chamber is maintained at 95%, and this cycle is repeated for 10 days.

[0089] The inoculation method for the large quantity of Phytophthora is as follows: using spores at a concentration of 2 × 10⁻⁶. 4 Spray two leaves of grafted seedlings with a Phytophthora suspension of 10 mL / mL, with a spraying amount of 10 mL per seedling.

[0090] S3. Repeat step S2 twice with the tuber 2 to harvest the tuber, which is the desired new potato germplasm with high resistance to late blight.

[0091] The severity grading criteria for late blight are as follows: Grade 0: No lesions on any leaves; Grade 1: A few lesions on a few leaves; Grade 3: Lesions on less than 1 / 4 of the leaves, or a few small lesions on the upper stems; Grade 5: Lesions on 1 / 4 to 1 / 2 of the leaves, or typical lesions on the upper stems; Grade 7: Lesions on more than 1 / 2 of the leaves, or typical lesions on the upper stems; Grade 9: Lesions on almost all leaves, or most leaves wither and die, and even the stems may die.

[0092] Example 2: Selecting potato materials with high resistance to late blight at grade 0 (e.g., Figure 5 Example 3: Grade 1 potato material resistant to late blight was selected (e.g., ...). Figure 6 This indicates that the method of the present invention can breed potato materials resistant to late blight, and the germplasm materials bred by grafting with late blight-resistant tomatoes as rootstock and potatoes as scions have stronger resistance to late blight.

[0093] Comparative Example 1: The method of Comparative Example 1 is similar to that of Example 2, except that the step of "(4) screening for late blight resistance germplasm" is omitted. The first batch of potato materials harvested after grafting is repeatedly propagated for one generation, and then winter-planted in the field under natural light and temperature conditions for late blight resistance testing. The results show that the plant growth is as follows. Figure 7 As shown, the late blight incidence rate of these potato materials reached 45%, classified as level 1 and level 3, with level 3 accounting for approximately 40%. This is compared to potato materials obtained after late blight resistance germplasm screening. Figure 5 The resistance to late blight varied considerably among the grafted potato materials, but all showed stronger resistance than the potato material Feuri used as the scion during field growth when late blight broke out (the incidence of disease in plants generally exceeded 80%). This was mainly because the late blight resistance genes from tomatoes were fully expressed and transmitted after the S1 and S2 stress treatments in step (4), thus significantly improving the late blight resistance of Example 2. That is, the potato material obtained after grafting tomatoes and potatoes can further improve its resistance to late blight by subjecting it to sodium chloride stress + Phytophthora stress + high temperature and high humidity stress, and make the late blight resistance of the potato material stable and heritable, which is beneficial for later production and use.

[0094] Comparative Example 2: The method of Comparative Example 2 is similar to that of Example 2, except that the S1 treatment in step (4) for screening germplasm resistant to late blight is free of sodium chloride stress. The potatoes bred as a result show some differences in resistance to late blight, with plants exhibiting late blight level 1 and level 3, and the incidence rate of level 3 plants being approximately 10%.

[0095] Comparative Example 3: The method of Comparative Example 3 is similar to that of Example 2, except that in step (4) screening of late blight resistant germplasm, step S2 is omitted, i.e., no large-scale inoculation with Phytophthora is performed, and no high temperature and high humidity treatment steps are used. As a result, the potatoes bred in this way have certain differences in resistance to late blight, with not only plants of late blight grade 1 and grade 3 appearing, but also grade 3 plants having an incidence rate of 26%.

[0096] Comparative Example 4: The method of Comparative Example 4 is similar to that of Example 2, except that in step (4) late blight resistance germplasm screening, S2, there is no high temperature and high humidity treatment step. The potatoes bred as a result show certain differences in resistance to late blight, with plants exhibiting late blight level 1 and level 3, and the incidence rate of level 3 plants being approximately 17%.

[0097] Comparative Example 5: The method in Comparative Example 5 is similar to that in Example 2, except that a conventional potato aeroponic device is used for aeroponic management of the grafted seedlings. Specifically, the potato aeroponic device does not have a ring-shaped rubber pad, and the nozzles of the second spray system spray the nutrient solution directly upwards from the bottom of the box. As a result, the grafting survival rate is only 65%. In contrast, the grafted seedlings cultivated using the potato aeroponic device in Example 1 have a survival rate of over 95%.

[0098] In summary, this invention grafts late blight-resistant tomato seedlings with 3-4 compound leaves onto late blight-resistant potato seedlings with 4-5 compound leaves. After grafting, a self-made aeroponic device is used for aeroponic management, and late blight-resistant germplasm is screened to breed the desired new late blight-resistant potato germplasm. During the late blight-resistant germplasm screening, grafted seedling 1 is first subjected to sodium chloride stress, which increases the expression level of related resistance genes in grafted seedling 1. Then, a small amount of Phytophthora inoculated is applied. Under the stimulation of a small amount of Phytophthora inoculated, grafted seedling 1 has stronger self-defense and a higher expression level of related resistance genes. Through the transfer between rootstock and scion, plants with certain disease resistance can be preliminarily screened. Grafted seedling 2 is inoculated with a large amount of Phytophthora inoculated and subjected to high temperature and high humidity treatment, which can promote the expression and transfer of disease resistance-related genes. Simultaneously, the harvested potato materials underwent multiple generations of repeated grafting and continuous screening, ensuring the full expression and stabilization of genes from late blight-resistant tomatoes. This ultimately led to the breeding of new, stable late blight-resistant potato germplasm, beneficial for production applications. Furthermore, the aeroponic device, with its ring-shaped rubber pads on the planting board, reduces contact between the grafting wound and moisture from the container, minimizing disease and rot, and improving the survival rate of grafted seedlings. The nutrient solution spray nozzles extend approximately 15 cm from the bottom of the planting board at a 30-45 degree angle to the container. This design avoids direct spraying from below, which could easily enter the planting holes on the planting board and contaminate the grafting interface. It also allows for closer, more even spraying onto the roots, enabling more efficient nutrient absorption, promoting faster healing of the grafting interface, and facilitating plant growth.

[0099] The foregoing description of specific exemplary embodiments of the present invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is obvious that many changes and variations can be made based on the above teachings. Although embodiments of the invention have been shown and described, these specific embodiments are merely explanations of the invention and are not intended to limit it. The specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. The purpose of selecting and describing exemplary embodiments is to explain the specific principles of the invention and its practical application, so that those skilled in the art, after reading this specification, can make modifications, substitutions, variations, and various choices and changes to the embodiments as needed without departing from the principles and spirit of the invention, provided that such modifications, substitutions, variations, and choices and changes are within the scope of the claims of the invention and are protected by patent law.

Claims

1. A method for creating new potato germplasm highly resistant to late blight through interspecific grafting, characterized in that, Includes the following steps: (1) Cultivation of potato seedlings and tomato seedlings: After the seeds of late blight-resistant tomatoes are pre-germinated, they are sown in seedling trays containing seedling substrate. When the seeds have grown to two compound leaves, they are transferred to a self-made aeroponic device and continued to grow until they have three to four compound leaves. Conventional aeroponic management is then used to cultivate the plants. Select virus-free potato seed tubers that have already sprouted, have good market demand, but are not resistant to or have low resistance to late blight, and plant them in flowerpots with seedling substrate. Under natural light, cultivate them until they grow to 4-5 compound leaves. Then cut them at the joint of the seed tuber, keeping 2-3 compound leaves, soak them in rooting solution for 25-35 minutes, and transplant them into a self-made aeroponic device. Before they have rooted, only spray them with rooting solution containing rooting powder. When the roots are 1-2 cm long, start spraying nutrient solution and carry out conventional aeroponic management to cultivate the plants. (2) Grafting with each other as rootstock and scion: Use potatoes with good marketability and high market demand but not resistant or low resistant to late blight as rootstock and tomatoes resistant to late blight as scions, or use tomatoes resistant to late blight as rootstock and potatoes with good marketability and high market demand but not resistant or low resistant to late blight as scions. The grafting operation includes: the method is cleft grafting, which is to cut the rootstock stem at a thick part under shaded conditions and split it in the middle with a 1-2cm opening. Then, cut off the lower end of the scion of similar thickness, retaining 1-2 functional leaves. Cut the scion stem segment into a gentle slope, keeping both sides symmetrical and the length equal to the opening of the rootstock. Then insert the scion into the rootstock so that the cut surface of the rootstock and the cut surface of the scion are in close contact, and fix it with a grafting clip. (3) Treatment of grafted plants: Grafted tomato seedlings resistant to late blight / potato seedlings that are not resistant or have low resistance to late blight, or potato seedlings that are not resistant or have low resistance to late blight / tomato seedlings that are resistant to late blight, are further cultivated in an aeroponic device. The ambient temperature of the aeroponic device is controlled at 20-24℃ and the relative humidity at 60-70% to prevent the grafted seedlings from dehydrating and dying. In the first week, the seedlings are covered with a shade net with a shading rate of 70-80%, and then exposed to natural light for 3-4 weeks. During this period, all the runners are cut off to obtain 1 grafted seedling. (4) Screening of new germplasm with high resistance to late blight: S1. First, the grafted seedling 1 is subjected to sodium chloride stress, then a small amount of Phytophthora inoculated, and plants with certain disease resistance are selected and moved outdoors for cultivation. The tubers are harvested in time to obtain tuber 1. The sodium chloride stress is achieved by spraying one leaf of the grafted seedling 1-2 times daily for 2-3 consecutive days with a sodium chloride solution of 100-150 mmol / L, spraying until moist each time; the inoculation with a small amount of Phytophthora refers to using a spore concentration of 2×10⁻⁶. 4 Spray one leaf of grafted seedling with a Phytophthora suspension of 1-2 mL per plant. S2. After cultivating the tuber 1 and the late blight resistant tomato according to step (1) and grafting them as rootstock and scion according to step (2), the grafted seedling 2 is obtained by managing them according to step (3). After inoculating the grafted seedling 2 with a large amount of Phytophthora, the seedling is subjected to high temperature and high humidity treatment. Plants with high resistance to late blight are selected and moved outdoors for cultivation. The tubers are harvested in time to obtain tuber 2. The high temperature and high humidity treatment is as follows: first, adjust the temperature to 26-30℃ and incubate under light for 14-16 hours, then adjust the temperature to 18-22℃ and incubate in the dark for 8-10 hours. The humidity is maintained at 90-95% throughout the process, and this cycle is repeated for 7-10 days. S3. Repeat step S2 1-2 times with the tuber 2 to harvest the tuber, which is the desired new potato germplasm with high resistance to late blight.

2. The method for creating new potato germplasm highly resistant to late blight through interspecific grafting according to claim 1, characterized in that, In step (1), the aeroponic device includes: a first aeroponic box, a second aeroponic box, a first spray system, a second spray system, a planting plate, and an annular rubber pad; The first aeroponic box is positioned above the second aeroponic box, and the first and second aeroponic boxes are connected by the planting plate. The top of the first aeroponic box is open. The first spray system is located inside the first aeroponic box, and the second spray system is located inside the second aeroponic box. Planting holes are spaced apart on the planting plate, and an annular rubber gasket is positioned above the planting plate, corresponding vertically to the planting holes. The annular rubber gasket includes a first gasket and a second gasket stacked together, with the first gasket positioned above the second gasket. The inner diameter of the first gasket is smaller than the inner diameter of the second gasket, and the inner circles of the first and second gaskets are provided with easy-tear openings.

3. The method for creating new potato germplasm highly resistant to late blight through interspecific grafting according to claim 2, characterized in that, The annular rubber gasket also includes a third gasket, which is stacked between the first gasket and the second gasket. The inner diameter of the third gasket is larger than the inner diameter of the first gasket and smaller than the inner diameter of the second gasket. The inner circle of the third gasket is also provided with an easy-tear opening.

4. The method for creating new potato germplasm highly resistant to late blight through interspecific grafting according to claim 2, characterized in that, The nozzle of the second spraying system extends 10-20 cm downwards at a 30-45 degree angle from the bottom of the planting board and the junction of the second aeroponic box to spray.

5. The method for creating new potato germplasm highly resistant to late blight through interspecific grafting according to claim 1, characterized in that, In step (4) S1, the plant with certain disease resistance refers to a plant with one of the following diseases: the disease is that the whole plant leaves have no disease spots, a few individual leaves have individual disease spots, or less than 1 / 4 of the whole plant leaves have disease spots.

6. The method for creating new potato germplasm highly resistant to late blight through interspecific grafting according to claim 1, characterized in that, In step (4) S2, the inoculation method for the large quantity of Phytophthora is: using a spore concentration of 2×10 4 Spray two leaves of grafted seedlings with a Phytophthora suspension of 5-10 mL per plant.

7. The method for creating new potato germplasm highly resistant to late blight through interspecific grafting according to claim 1, characterized in that, In step (4) S2, the plant with high resistance to late blight refers to a plant with no disease spots on all leaves or with individual disease spots on individual leaves.

8. The method for creating new potato germplasm highly resistant to late blight through interspecific grafting according to claim 2, characterized in that, The aeroponic device is placed in a temperature-controlled air-conditioned room for temperature and humidity control, and / or, a temperature control device, a light control device, and a humidity sensor are provided in the first aeroponic chamber. The temperature control device includes a temperature probe, a heating element, and a temperature controller. The temperature probe and the heating element are electrically connected to the temperature controller, which controls the temperature inside the first aeroponic chamber. The light control device includes an LED light and a timer. The LED light and the timer are connected, and the timer controls the illumination time inside the first aeroponic chamber. The humidity sensor is connected to the first spray system to control the air humidity inside the first aeroponic chamber.

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