Method and device for creating new potato germplasm with high late blight resistance through interspecific grafting

Abstract

The invention discloses a method and a device for creating a new potato germplasm with high late blight resistance through interspecific grafting. The method comprises the following steps: (1) cultivating potato seedlings and late blight-resistant tomato seedlings; (2) mutual stock-scion grafting; (3) treating grafted plants to obtain grafted seedlings 1; (4) screening germplasm with high late blight resistance: S1, performing sodium chloride stress on the grafted seedlings 1, inoculating a small amount of phytophthora, screening out plants with certain disease resistance, transferring the plants to outdoor culture, and harvesting tubers at proper time to obtain tubers 1; s2, the tubers 1 are planted and then grafted with tomatoes to obtain grafted seedlings 2, the grafted seedlings 2 are inoculated with a large number of phytophthora and then subjected to high-temperature and high-humidity treatment, plants with high disease resistance are screened out and moved outdoors to be cultured, tubers are harvested in good time, and tubers 2 are obtained; and S3, repeating the step S2 for 1-2 times on the tubers 2, and harvesting tubers, namely the required new potato germplasm with high late blight resistance. The new potato germplasm with high late blight resistance and genetic stability is obtained, the variety breeding time is shortened, and the cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of breeding, and particularly relates to a method and a device for creating new potato germplasm with high resistance to late blight by interspecific grafting. Background Art

[0002] Potato late blight has a large degree of damage and covers a wide area, which can lead to a large-scale reduction in potato production or even a total crop failure, causing serious economic losses. Late blight mainly occurs under high temperature and high humidity environmental conditions. For example, in my country's winter potato cropping areas, it mainly occurs during the rainy season from February to April when the temperature rises, and in potato primary and secondary cropping areas, it occurs after the rain from May to September. Late blight mainly harms potato petioles, stems and tubers, but the most susceptible parts are leaves. When the leaves are infected by pathogens, small dark green spots will first appear on the leaf tips or edges. Under humid conditions, the spots expand rapidly, showing irregular dark green water-soaked colors. When the petioles and stems are infected by pathogens, the leaves wither and droop, and finally the entire plant becomes charred and wet and rotten until the stems fall over and die.

[0003] Compared with chemical control and field management control, the selection and application of disease-resistant varieties is the most effective and safe way to prevent potato late blight. At present, late blight-resistant varieties are mainly cultivated through hybrid breeding, selective breeding, mutation breeding and genetic engineering, but whether it is hybrid breeding, selective breeding or mutation breeding, it takes a long time and it is difficult to meet production needs. Grafting is to connect the incisions of two plants together to heal them into a new combined plant. Grafting technology in agriculture is mainly used to increase yield and stress resistance. Many studies have shown that there is material exchange in the plant body after grafting. Plant hormones, small molecule metabolites, inorganic salts, sugars, proteins, DNA and RNA can be transferred between rootstock and scion. Therefore, grafting can change the gene expression of plants. There are patents that disclose the use of grafting technology to control potato late blight, such as the patent "A method for controlling potato late blight using grafting technology" (CN 109156188 B). This method uses potato varieties with high resistance to late blight as scions and potato varieties with low resistance to late blight that have good commerciality and large market demand as rootstocks. After grafting, no chemical agents need to be applied during the entire growth period of potatoes, achieving biological control of potato late blight and achieving green and pollution-free production. However, this grafting method can only be applied in the production season, and cannot obtain heritable potato germplasm resistant to late blight for production for many years. It cannot solve the problems of the urgent need for stable late blight-resistant varieties in potato production and the long breeding time and high cost of varieties.

[0004] On the other hand, the existing potato aeroponic device generally places a 2-3 cm thick foam board on the top of the aeroponic box, and a planting hole is opened in the board to plant the plants. After the potato and tomato are grafted, the grafting part is generally fixed by a fixing clamp, and then the fixing clamp is set on the planting hole, so that the grafting part is above the foam board and does not fall into the box body and is contaminated by the nutrient solution and rots. However, in the early stage of grafting, the grafting part has not healed yet and has wounds, which is very easy to be contaminated by the nutrient solution directly sprayed from the bottom of the box through the planting hole and become sick and rotten, reducing the survival rate of the grafted seedlings. Summary of the invention

[0005] The purpose of the present invention is to provide a method and device for creating new potato germplasm with high resistance to late blight by interspecific grafting, so as to solve the problems that there is no technology for creating potato germplasm with resistance to late blight by grafting and the conventional variety breeding time is too long and the cost is too high. The specific technical scheme is as follows:

[0006] A method for creating new potato germplasm with high resistance to late blight by interspecific grafting comprises the following steps:

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

[0008] After germination treatment, seeds of late blight-resistant tomatoes were sown in plug trays filled with seedling medium. When they grew to 2 compound leaves, they were transferred to a homemade aeroponic device and continued to be cultivated until they grew to 3-4 compound leaves. Conventional aeroponic management was used to cultivate the plants.

[0009] Select germinated virus-free potato seed potatoes with good commercial value and large market demand but low or no resistance to late blight and plant them in a flower pot covered with a seedling medium. Cultivate them under natural light conditions until they grow to 4-5 compound leaves. Then cut them from the connection of the seed potatoes, retain 2-3 compound leaves, soak them in rooting liquid for 25-35 minutes, and transplant them to a self-made aeroponic device. Before they take root, only spray the rooting liquid with rooting powder. When the roots grow to 1-2 cm long, start spraying nutrient solution and carry out conventional aeroponic management to cultivate the plants.

[0010] (2) Mutual rootstock-scion grafting: using potatoes with good commercial quality and high market demand but low or no resistance to late blight as rootstocks and tomatoes with resistance to late blight as scions; or using tomatoes with resistance to late blight as rootstocks and potatoes with good commercial quality and high market demand but low or no resistance to late blight as scions;

[0011] The grafting operation includes: the method is a cleft grafting method, that is, the stock stem is cut at the thick part under shading conditions, and a 1-2 cm opening is split from the middle, then the lower end of the scion with similar thickness is cut off, 1-2 functional leaves are retained, the scion stem segment is cut into a gentle slope, the two sides are kept symmetrical and the length is equal to the stock opening, and then the scion is inserted into the stock so that the stock cut surface is closely fitted with the scion cut surface, and fixed with a grafting clamp;

[0012] (3) Treatment of grafted plants: Grafted late blight-resistant tomato / potato (scion / stock) grafted seedlings with no or low resistance to late blight, or potato (scion / stock) grafted seedlings with no or low resistance to late blight / tomato grafted seedlings with no or low resistance to late blight are continued to be cultivated in an aeroponic device, and the ambient temperature of the aeroponic device is controlled at 20-24° C. and the relative humidity is 60-70% to prevent the grafted seedlings from dehydration and death. In the first week, the grafted seedlings are covered with a sunshade net with a shading rate of 70-80%, and then exposed to natural light for another 3-4 weeks. During this period, all long runners are cut off to obtain grafted seedling 1;

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

[0014] S1, first subjecting the grafted seedlings 1 to sodium chloride stress, then inoculating a small amount of Phytophthora, selecting plants with certain disease resistance, moving them to outdoor cultivation, and harvesting tubers in due time to obtain tubers 1;

[0015] S2, cultivating the tuber 1 and the late blight-resistant tomato according to step (1), grafting them as stock and scion according to step (2), and managing them according to step (3) to obtain grafted seedlings 2, inoculating a large amount of Phytophthora into the grafted seedlings 2, subjecting them to high temperature and high humidity treatment, selecting plants with high resistance to late blight, moving them to outdoor cultivation, and harvesting the tubers in due time to obtain tubers 2;

[0016] The high temperature and high humidity treatment is as follows: firstly, the temperature is adjusted to 26-30°C, and the light is cultured for 14-16 hours, then the temperature is adjusted to 18-22°C, and the dark is cultured for 8-10 hours, and the humidity is maintained at 90-95% during the whole process, and this cycle is repeated for 7-10 days;

[0017] S3. Repeat step S2 for 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 comprises: a first aeroponic box, a second aeroponic box, a first spray system, a second spray system, a planting plate and an annular rubber gasket;

[0019] The first aerosol box is arranged above the second aerosol box, and the first aerosol box and the second aerosol box are connected through the planting plate; the top of the first aerosol box is set as an opening; the first spray system is arranged in the first aerosol box, and the second spray system is arranged in the second aerosol box; planting holes are arranged at intervals on the planting plate, and the annular rubber gasket is arranged above the planting plate and corresponds to the position of the planting holes up and down; the annular rubber gasket includes a stacked first gasket and a second gasket, the first gasket is arranged 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 gasket and the second gasket 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 notch.

[0021] Furthermore, the nozzle of the second spray system extends 10-20 cm downward from the junction of the bottom of the planting plate and the second mist culture box at an angle of 30-45 degrees for spraying.

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

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

[0024] Furthermore, in S1 of step (4), the plant with a certain disease resistance refers to a plant having one of the following conditions: the plant has no disease spots on all leaves, individual leaves have individual disease spots, and less than 1 / 4 of the leaves of the plant have disease spots.

[0025] Furthermore, in step (4) S2, the method of inoculating a large amount of Phytophthora is as follows: using a spore concentration of 2×10 4 Spray 2 leaves of the grafted seedlings with a suspension of Phytophthora spp. / mL, with a spraying amount of 5-10 mL per plant.

[0026] Furthermore, in S2 of step (4), 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 mist cultivation device is placed in a temperature-controllable 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 mist cultivation box, the temperature control device comprises a temperature probe, a heating tube and a temperature controller, the temperature probe and the heating tube are electrically connected to the temperature controller respectively, and the temperature in the first mist cultivation box is controlled by the temperature controller; the light control device comprises an LED lamp and a time controller, the LED lamp and the time controller are connected, and the illumination time in the first mist cultivation box is controlled by the time controller; the humidity sensor is connected to the first spray system to control the air humidity in the first mist cultivation box.

[0028] Compared with the existing technology, the present invention has the following beneficial effects:

[0029] 1. The invention grafts late blight-resistant tomato seedlings grown to 3-4 compound leaves with potato seedlings grown to 4-5 compound leaves with good commercial quality and large market demand but no or low resistance to late blight, adopts a self-made aeroponic device for aeroponic management after grafting, and carries out late blight-resistant germplasm screening, thereby breeding required new potato germplasm with high late blight resistance, and solves the problems that there is no technology for creating new potato germplasm with late blight resistance by grafting method at present, and conventional variety breeding time is too long and costly.

[0030] 2. The mist cultivation device of the present invention can reduce the contact between the grafting wound and the water vapor coming out of the box body by providing an annular rubber gasket on the planting plate, reduce the phenomenon of disease and rot, and improve the survival rate of the grafted seedlings; the nutrient solution nozzle extends downward about 15 cm from the joint of the bottom of the planting plate and the box body at an angle of 30-45 degrees. On the one hand, it prevents the nozzle from directly spraying into the planting hole on the planting plate from below and causing contamination of the grafting port. On the other hand, it can spray at a close distance and spray more evenly on the roots, so that the roots can absorb nutrients more efficiently, promote faster healing of the grafting port and plant growth.

[0031] 3. When the present invention screens new potato germplasm with high resistance to late blight, the grafted seedling 1 is first subjected to sodium chloride stress, which can increase the expression of the relevant resistance genes of the grafted seedling 1, and then a small amount of Phytophthora is inoculated. Under the stimulation of a small amount of Phytophthora, the self-defense of the grafted seedling 1 is stronger, and the expression of the relevant resistance genes is greater, which accelerates the transfer of the relevant genes between the rootstock and the scion, and can preliminarily screen out plants with certain disease resistance; a large amount of Phytophthora is inoculated into the grafted seedling 2 and subjected to high temperature and high humidity treatment, creating a late blight outbreak environment, which can further promote the expression and transfer of the relevant genes with disease resistance. At the same time, the harvested potato materials are continuously grafted and screened for multiple generations, so that the genes from the late blight-resistant tomatoes are more fully expressed, quickly transferred and stabilized, and finally a new potato germplasm with high resistance to late blight with stable disease resistance traits is selected, which is more conducive to production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

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

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

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

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

[0037] Figure 5 This is the new germplasm material created in Example 2, and the late blight disease grade shown in the field is grade 0.

[0038] Figure 6 This is the new germplasm material created in Example 3, and the late blight disease grade shown in the field is grade 1.

[0039] Figure 7 For the new germplasm material created in comparative example 1, the late blight disease grade in the field was grade 3.

[0040] Among them, the main reference numerals are described as follows:

[0041] 1. First mist culture box; 2. Second mist culture box; 3. Planting plate; 31. Planting hole; 4. Annular rubber gasket; 41. First gasket; 42. Second gasket; 43. Third gasket; 44. Tear-off; 5. Temperature control device; 51. Temperature probe; 52. Heating tube; 53. Temperature controller; 6. Light control device; 61. LED lamp; 62. Time controller; 7. First spray system; 8. Second spray system; 81. Nozzle; 9. Shade curtain; 10. Collection door; 11. Humidity sensor. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] In the description of the present invention, it should be noted that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "top", "bottom", "top surface", "bottom surface", "inside", "outside", "inside", "outside" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, which are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention.

[0044] In the description of the present invention, the meaning of "several" is one or more, the meaning of "more" is more than two, "greater than", "less than", "exceed" etc. are understood to exclude the number itself, and "above", "below", "within" etc. are understood to include the number itself. If there are terms "first", "second" and "third" in the description, they are only used for the purpose of description and distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0045] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected" and "set" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal connection of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. The following is a description of an embodiment of the present invention based on its overall structure.

[0046] Example 1

[0047] Combination Figure 1-2 , an aeroponic device, comprising: a first aeroponic box, a second aeroponic box, a first spray system, a second spray system, a planting plate, and an annular rubber gasket; the first aeroponic box is arranged above the second aeroponic box, and the first aeroponic box and the second aeroponic box are connected through the planting plate;

[0048] The first aerosol cultivation box is provided 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 in the first aerosol cultivation box; the temperature control device is used to control the temperature in the first aerosol cultivation box; the light control device is used to control the light in the first aerosol cultivation box; the humidity sensor is used to sense the humidity in the first aerosol cultivation box; the top of the first aerosol cultivation box is set as an opening, and when it is necessary to spray sodium chloride solution or phytophthora suspension, it can be operated from the opening;

[0049] The second aeroponic box is provided with a second spray system, which is provided at four corners and is used to regularly spray water or nutrient solution to the roots and tubers of the plants in the second aeroponic box; the second aeroponic box is also provided with a shading curtain, a collection door and other structures;

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

[0051] The annular rubber gasket is arranged above the planting plate and corresponds to the position of the planting hole in the upper and lower parts; the annular rubber gasket is used to block the water vapor from the second aerosol box and fix the crops on the planting plate.

[0052] Specific, combined Figure 3-4 , the annular rubber gasket includes a stacked first gasket, a second gasket and a third gasket, the first gasket is arranged 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 gasket and the second gasket are provided with an easy-tear notch. The third gasket 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, and the inner circle of the third gasket is also provided with an easy-tear notch. The annular rubber gasket is made of rubber and has a certain elasticity, which can adapt to the growth of crops. The setting of the easy-tear notch allows the gasket to be taken off according to the growth of the crop stems. Preferably, the easy-tear notches of the first gasket, the second gasket and the third gasket are staggered.

[0053] Combination Figure 2 The nozzle of the second spray system extends downward 10-20 cm from the junction of the bottom of the planting board and the second mist culture box at an angle of 30-45 degrees to spray. On the one hand, it prevents the nozzle from spraying directly from below into the planting hole on the planting board and causing contamination of the grafting point. On the other hand, it can spray at a close distance and spray more evenly on the roots, allowing the roots to absorb nutrients more efficiently, promoting faster healing of the grafting point 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 respectively, and the temperature in the first mist culture box is controlled by the temperature controller.

[0055] Furthermore, the light control device includes an LED lamp and a time controller, the LED lamp and the time controller are electrically connected, and the lighting time in the first mist cultivation box is controlled by the time controller.

[0056] Furthermore, the humidity sensor is connected to the first spray system to control the air humidity in the first aerosol culture box.

[0057] In addition, in another embodiment, the first mist culture box is also replaced by a support frame. When it is set as a support frame, the temperature, light and humidity of the stems and leaves of the plants are all uniformly controlled by the equipment in the air-conditioned room.

[0058] The mist culture device of the present invention is used to cultivate grafted potato and tomato seedlings. By providing an annular rubber gasket on the planting plate, the water vapor from the second mist culture box can be blocked and the grafting wound can be raised, so as to reduce the contact between the grafting wound and the water vapor coming out of the box body, reduce the phenomenon of disease and rot, and improve the survival rate of the grafted seedlings. The nutrient solution nozzle extends downward by about 15 cm from the joint of the bottom of the planting plate and the box body at an angle of 30-45 degrees. On the one hand, it is prevented from spraying directly from below into the planting hole on the planting plate to cause contamination of the grafting port. On the other hand, it can spray at a close distance and spray more evenly on the roots, so that the roots can absorb nutrients more efficiently, promote faster healing of the grafting port and plant growth.

[0059] Example 2

[0060] Material cultivation and grafting were carried out in the potato aeroponic room of the Institute of Economic Crops of Guangxi Academy of Agricultural Sciences, with natural light, temperature of 20°C and relative humidity of 60%. The potato aeroponic room was equipped with a plurality of aeroponic devices described in Example 1. The potato material was "Feureta" which has good commercial value but is not resistant to late blight, and the tomato material resistant to late blight was 2A37. "Feureta" can be purchased from Guangxi Academy of Agricultural Sciences or the general market, and 2A37 tomatoes are varieties bred by Guangxi University. Phytophthora fungi originated from the Institute of Microbiology of Guangxi Academy of Agricultural Sciences.

[0061] A method for creating new potato germplasm with high resistance to late blight by interspecific grafting comprises the following steps:

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

[0063] After germination treatment, 2A37 tomato seeds were sown in a plug tray filled with a seedling medium. When they grew to 2 compound leaves, they were transferred to a homemade aeroponic device and continued to be cultivated until they grew to 3-4 compound leaves. Conventional aeroponic management was used to cultivate the plants.

[0064] Select the germinated virus-free "Feureta" potato seed potatoes and plant them in a flower pot covered with a seedling medium. Cultivate them under natural light until they grow to 4-5 compound leaves. Then cut them from the connection of the seed potatoes, retain 2-3 compound leaves, soak them in rooting liquid for 25 minutes, and transplant them into a homemade aeroponic device. Before they take root, only spray the rooting liquid with rooting powder. When the roots grow to 1-2 cm long, start spraying nutrient solution and carry out conventional aeroponic management to cultivate the plants.

[0065] (2) Mutual rootstock-scion grafting: 2A37 tomato is used as the rootstock and “Feureta” as the scion; the grafting time is selected within one week before New Year’s Day;

[0066] The grafting operation includes: the method is a cleft grafting method, that is, the stock stem is cut at the thick part under shading conditions, and a 1-2 cm opening is split from the middle, then the lower end of the scion with similar thickness is cut off, 1-2 functional leaves are retained, the scion stem segment is cut into a gentle slope, the two sides are kept symmetrical and the length is equal to the stock opening, and then the scion is inserted into the stock so that the stock cut surface is closely fitted with the scion cut surface, and fixed with a grafting clamp;

[0067] (3) Treatment of grafted plants: The grafted "Feureta" / 2A37 tomato (scion / stock) seedlings were further cultivated in the aeroponic device, and the ambient temperature of the aeroponic device was controlled at 20°C and the relative humidity was 60% to prevent the grafted seedlings from dehydration and death. In the first week, they were covered with a sunshade net with a shading rate of 70%, and then exposed to natural light for another 4 weeks. During this period, all the long runners were cut off to obtain grafted seedling 1;

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

[0069] S1, first subjecting the grafted seedlings 1 to sodium chloride stress, then inoculating a small amount of Phytophthora, selecting plants with certain disease resistance, moving them to outdoor cultivation, and harvesting tubers in due time to obtain tubers 1; the plants with certain disease resistance refer to plants with one of the following conditions, the conditions being plants with no disease spots on leaves of the whole plant, plants with individual disease spots on individual leaves, and plants with disease spots on less than 1 / 4 of leaves of the whole plant;

[0070] The sodium chloride stress is to spray one leaf of the grafted seedling with a sodium chloride solution of 100 mmol / L once a day for 2 consecutive days, and spray until the leaves are moistened each time; the inoculation of a small amount of Phytophthora means: using a spore concentration of 2×10 4 Spray one leaf of the grafted seedlings with a suspension of Phytophthora spp. / mL, with a spraying amount of 1 mL per plant.

[0071] S2, cultivating the tuber 1 and the 2A37 tomato according to step (1), grafting them as rootstocks and scions according to step (2), and managing them according to step (3) to obtain grafted seedlings 2, inoculating a large amount of Phytophthora into the grafted seedlings 2, and subjecting them to high temperature and high humidity treatment, selecting plants with no disease spots on the leaves of the whole plant or with individual disease spots on individual leaves, moving them to outdoor cultivation, and harvesting the tubers in due time to obtain tubers 2;

[0072] The high temperature and high humidity treatment is as follows: firstly, the temperature of the first mist culture box is adjusted to 26°C, and the light culture is carried out for 14 hours, and then the temperature is adjusted to 18°C, and the dark culture is carried out for 10 hours. During the whole process, the humidity of the first mist culture box is maintained at 90%, and this cycle is repeated for 7 days;

[0073] The method of inoculating a large number of Phytophthora is as follows: using a spore concentration of 2×10 4 Spray 2 leaves of the grafted seedlings with a suspension of Phytophthora spp. / mL, with a spraying amount of 5 mL per plant.

[0074] S3. Repeat step S2 once with the tuber 2 to 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 room of the Institute of Economic Crops of Guangxi Academy of Agricultural Sciences, with natural light, temperature of 24°C and relative humidity of 70%. The potato aeroponic room was equipped with a plurality of aeroponic devices described in Example 1. The potato material was "Feureta" which has good commercial value but is not resistant to late blight, and the tomato material resistant to late blight was 2A37. "Feureta" can be purchased from Guangxi Academy of Agricultural Sciences or the general market, and 2A37 tomatoes are varieties bred by Guangxi University. Phytophthora came from the Institute of Microbiology of Guangxi Academy of Agricultural Sciences.

[0077] A method for creating new potato germplasm with high resistance to late blight by interspecific grafting comprises the following steps:

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

[0079] After germination treatment, 2A37 tomato seeds were sown in a plug tray filled with a seedling medium. When they grew to 2 compound leaves, they were transferred to a homemade aeroponic device and continued to be cultivated until they grew to 3-4 compound leaves. Conventional aeroponic management was used to cultivate the plants.

[0080] Select the germinated virus-free "Feureta" seed potatoes and plant them in a pot covered with a seedling medium. Cultivate them under natural light until they grow to 4-5 compound leaves. Then cut them from the connection of the seed potatoes, retain 2-3 compound leaves, soak them in rooting liquid for 25-35 minutes, and transplant them into a homemade aeroponic device. Before they take root, only spray the rooting liquid with rooting powder. When the roots grow to 1-2 cm long, start spraying nutrient solution and carry out conventional aeroponic management to cultivate the plants.

[0081] (2) Mutual rootstock-scion grafting: use "Feureta" as the rootstock and 2A37 tomato as the scion; the grafting time should be within one week after New Year's Day;

[0082] The grafting operation includes: the method is a cleft grafting method, that is, the stock stem is cut at the thick part under shading conditions, and a 1-2 cm opening is split from the middle, then the lower end of the scion with similar thickness is cut off, 1-2 functional leaves are retained, the scion stem segment is cut into a gentle slope, the two sides are kept symmetrical and the length is equal to the stock opening, and then the scion is inserted into the stock so that the stock cut surface is closely fitted with the scion cut surface, and fixed with a grafting clamp;

[0083] (3) Treatment of grafted plants: The grafted 2A37 tomato / "Feureta" (scion / stock) seedlings were further cultivated in the aeroponic device, and the ambient temperature of the aeroponic device was controlled at 24°C and the relative humidity was 70% to prevent the grafted seedlings from dehydration and death. In the first week, they were covered with a sunshade net with a shading rate of 80%, and then exposed to natural light for another 3 weeks. During this period, all the long runners were cut off to obtain grafted seedling 1;

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

[0085] S1, first subjecting the grafted seedlings 1 to sodium chloride stress, then inoculating a small amount of Phytophthora, selecting plants with certain disease resistance, moving them to outdoor cultivation, and harvesting tubers in due time to obtain tubers 1; the plants with certain disease resistance refer to plants with one of the following conditions, the conditions being plants with no disease spots on leaves of the whole plant, plants with individual disease spots on individual leaves, and plants with disease spots on less than 1 / 4 of leaves of the whole plant;

[0086] The sodium chloride stress is to spray one leaf of the grafted seedling with a sodium chloride solution of 150 mmol / L twice a day for 3 consecutive days, and spray until the leaves are moistened each time; the inoculation of a small amount of Phytophthora means: using a spore concentration of 2×10 4 Spray one leaf of the grafted seedlings with a suspension of Phytophthora spp. / mL, with a spraying amount of 2 mL per plant.

[0087] S2, cultivating the tuber 1 and the 2A37 tomato according to step (1), grafting them as rootstocks and scions according to step (2), and managing them according to step (3) to obtain grafted seedlings 2, inoculating a large amount of Phytophthora into the grafted seedlings 2, and subjecting them to high temperature and high humidity treatment, selecting plants with no disease spots on the leaves of the whole plant or with individual disease spots on individual leaves, moving them to outdoor cultivation, and harvesting the tubers in due time to obtain tubers 2;

[0088] The high temperature and high humidity treatment is as follows: firstly, the temperature of the first mist culture box is adjusted to 30°C, and the light culture is carried out for 16 hours, and then the temperature is adjusted to 22°C, and the dark culture is carried out for 8 hours. During the whole process, the humidity of the first mist culture box is maintained at 95%, and this cycle is repeated for 10 days;

[0089] The method of inoculating a large number of Phytophthora is as follows: using a spore concentration of 2×10 4 Spray two leaves of the grafted seedlings with a suspension of Phytophthora spp. / mL, with a spraying amount of 10 mL per plant.

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

[0091] The late blight disease grading standard is: Level 0: no lesions on the leaves of the whole plant; Level 1: individual lesions on individual leaves; Level 3: less than 1 / 4 of the leaves of the whole plant have lesions, or there are individual small lesions on the upper stems of the plant; Level 5: 1 / 4 to 1 / 2 of the leaves of the whole plant have lesions, or there are typical lesions on the upper stems of the plant; Level 7: more than 1 / 2 of the leaves of the whole plant have lesions, or there are typical lesions on the upper stems of the plant; Level 9: almost all the leaves of the whole plant have lesions, or most of the leaves are dead, and even the stems are dead.

[0092] Example 2 Breeding of Grade 0 high late blight resistant potato materials (such as Figure 5 ); Example 3 bred a level 1 late blight resistant potato material (such as Figure 6 ), indicating that the method of the present invention can breed potato materials resistant to late blight, and the germplasm materials bred by using late blight-resistant tomatoes as rootstocks and potatoes as scions for grafting are more resistant to late blight.

[0093] Comparative Example 1: The method of Comparative Example 1 is similar to that of Example 2, except that the step "(4) screening of germplasm resistant to late blight" is omitted, and the first batch of potato materials harvested by grafting are repeatedly propagated for one generation, and then planted in the field under natural light and temperature conditions for late blight resistance testing. The results show that the growth of the plants is as follows: Figure 7 As shown in Figure 1, the incidence of late blight in these potato materials reached 45%, with grades 1 and 3, of which grade 3 accounted for about 40%. Compared with the potato materials obtained after late blight resistance screening ( Figure 5 ) have a large gap in late blight resistance, but they are all stronger than the potato material Feure used as the scion during grafting when the late blight breaks out during field growth (the diseased plants generally exceed 80%). It is mainly because the late blight resistance gene from tomato is fully expressed and transmitted after the stress treatment of S1 and S2 in step (4), so the late blight resistance of Example 2 is greatly improved. That is, the potato material obtained after tomato and potato grafting is further subjected to sodium chloride stress + Phytophthora stress + high temperature and high humidity stress, which can further improve the late blight resistance of the potato material and make the late blight resistance performance of the potato material stable and inherited, which is beneficial to the later production and use.

[0094] Comparative Example 2: The method of Comparative Example 2 is similar to that of Example 2, except that: S1 in step (4) of late blight resistance germplasm screening does not undergo sodium chloride stress treatment. The potatoes thus cultivated have certain differences in late blight resistance, with plants of late blight grade 1 and grade 3 appearing, and the incidence of grade 3 plants is about 10%.

[0095] Comparative Example 3: The method of Comparative Example 3 is similar to that of Example 2, except that: in step (4) of late blight resistance germplasm screening, there is no S2 step, that is, no large amount of Phytophthora inoculation, and no high temperature and high humidity treatment step. The potatoes thus cultivated have a certain difference in late blight resistance, not only plants with late blight grade 1 and grade 3 appear, but also the grade 3 plant has 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) of late blight resistance germplasm screening, S2, there is no high temperature and high humidity treatment step. The potatoes thus cultivated have certain differences in late blight resistance, and plants with late blight grade 1 and grade 3 appeared, and the incidence rate of plants with grade 3 was about 17%.

[0097] Comparative Example 5: The method of Comparative Example 5 is similar to that of Example 2, except that a conventional potato aeroponic device is used to manage the grafted seedlings. That is, the potato aeroponic device is not provided with an annular rubber gasket, and the nozzle of the second spray system is directed upward from the bottom of the box, and the nutrient solution is sprayed directly upward, resulting in a graft survival rate of only 65%. The survival rate of the grafted seedlings cultivated by the potato aeroponic device of Example 1 is as high as over 95%.

[0098] In summary, the present invention grafts late blight-resistant tomato seedlings that grow to 3-4 compound leaves with late blight-intolerant potato seedlings that grow to 4-5 compound leaves, and after grafting, a self-made aeroponic device is used for aeroponic management, and late blight-resistant germplasm screening is performed, so that the required late blight-resistant potato new germplasm can be bred. When screening the late blight-resistant germplasm, the grafted seedlings 1 are first subjected to sodium chloride stress, so that the expression amount of the relevant resistance gene of the grafted seedlings 1 can be increased, and then a small amount of phytophthora is inoculated. Under the stimulation of a small amount of phytophthora, the self-defense of the grafted seedlings 1 is stronger, and the expression amount of the relevant resistance gene is greater. Through the transmission between the rootstock and the scion, plants with certain disease resistance can be preliminarily screened out; and the grafted seedlings 2 are inoculated with a large amount of phytophthora and subjected to high temperature and high humidity treatment, so as to promote the expression and transmission of the relevant genes of the disease resistance. At the same time, the harvested potato materials are repeatedly grafted and continuously screened for multiple generations, so that the genes from the late blight-resistant tomatoes are fully expressed and stabilized, and finally a new late blight-resistant potato germplasm with stable traits is selected, which is conducive to production application. In addition, the mist culture device can reduce the contact between the grafting wound and the water vapor coming out of the box by providing an annular rubber gasket on the planting plate, reduce the phenomenon of disease and rot, and improve the survival rate of the grafted seedlings; the nutrient solution nozzle extends about 15 cm from the bottom of the planting plate and the junction of the box at 30-45 degrees. On the one hand, it prevents the nozzle from spraying directly from below into the planting hole on the planting plate and causing contamination of the grafting port. On the other hand, it can spray at a close distance and spray more evenly on the roots, so that the roots can absorb nutrients more efficiently, promote faster healing of the grafting port and plant growth.

[0099] The foregoing descriptions of specific exemplary embodiments of the present invention are for the purpose of explanation and illustration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is obvious that many changes and variations can be made based on the above teachings. Although an embodiment of the present invention has been shown and described, this specific embodiment is only an explanation of the present invention and is not a limitation of the invention. The specific features, structures, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can make modifications, substitutions, variations and various selections and changes to the embodiments without creative contribution as needed without departing from the principles and purpose of the present invention after reading this specification, but they are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for creating new potato germplasm with high resistance to late blight by interspecific grafting, characterized in that: The steps include: (1) Cultivation of potato and tomato seedlings: After germination treatment, seeds of late blight-resistant tomatoes were sown in plug trays filled with seedling medium. When they grew to 2 compound leaves, they were transferred to a homemade aeroponic device and continued to be cultivated until they grew to 3-4 compound leaves. Conventional aeroponic management was used to cultivate the plants. Select germinated virus-free potato seed potatoes with good commercial value and large market demand but low or no resistance to late blight and plant them in a flower pot covered with a seedling medium. Cultivate them under natural light conditions until they grow to 4-5 compound leaves. Then cut them from the connection of the seed potatoes, retain 2-3 compound leaves, soak them in rooting liquid for 25-35 minutes, and transplant them to a self-made aeroponic device. Before they take root, only spray the rooting liquid with rooting powder. When the roots grow to 1-2 cm long, start spraying nutrient solution and carry out conventional aeroponic management to cultivate the plants. (2) Mutual rootstock-scion grafting: using potatoes with good commercial quality and high market demand but low or no resistance to late blight as rootstocks and tomatoes with resistance to late blight as scions; or using tomatoes with resistance to late blight as rootstocks and potatoes with good commercial quality and high market demand but low or no resistance to late blight as scions; The grafting operation includes: the method is a cleft grafting method, that is, the stock stem is cut at the thick part under shading conditions, and a 1-2 cm opening is split from the middle, then the lower end of the scion with similar thickness is cut off, 1-2 functional leaves are retained, the scion stem segment is cut into a gentle slope, the two sides are kept symmetrical and the length is equal to the stock opening, and then the scion is inserted into the stock so that the stock cut surface is closely fitted with the scion cut surface, and fixed with a grafting clamp; (3) Treatment of grafted plants: Grafted late blight-resistant tomato / potato (scion / stock) grafted seedlings with no or low resistance to late blight, or potato (scion / stock) grafted seedlings with no or low resistance to late blight / tomato grafted seedlings with no or low resistance to late blight are continued to be cultivated in an aeroponic device, and the ambient temperature of the aeroponic device is controlled at 20-24° C. and the relative humidity is 60-70% to prevent the grafted seedlings from dehydration and death. In the first week, the grafted seedlings are covered with a sunshade net with a shading rate of 70-80%, and then exposed to natural light for another 3-4 weeks. During this period, all long runners are cut off to obtain grafted seedling 1; (4) Screening of new germplasm with high resistance to late blight: S1, first subjecting the grafted seedlings 1 to sodium chloride stress, then inoculating a small amount of Phytophthora, selecting plants with certain disease resistance, moving them to outdoor cultivation, and harvesting tubers in due time to obtain tubers 1; S2, cultivating the tuber 1 and the late blight-resistant tomato according to step (1), grafting them as stock and scion according to step (2), and managing them according to step (3) to obtain grafted seedlings 2, inoculating a large amount of Phytophthora into the grafted seedlings 2, subjecting them to high temperature and high humidity treatment, selecting plants with high resistance to late blight, moving them to outdoor cultivation, and harvesting the tubers in due time to obtain tubers 2; The high temperature and high humidity treatment is as follows: firstly, the temperature is adjusted to 26-30°C, and the light is cultured for 14-16 hours, then the temperature is adjusted to 18-22°C, and the dark is cultured for 8-10 hours, and the humidity is maintained at 90-95% during the whole process, and this cycle is repeated for 7-10 days; S3. Repeat step S2 for 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 with high resistance to late blight by interspecific grafting according to claim 1, characterized in that: In step (1), the aeroponic device comprises: a first aeroponic box, a second aeroponic box, a first spray system, a second spray system, a planting plate and an annular rubber gasket; The first aerosol box is arranged above the second aerosol box, and the first aerosol box and the second aerosol box are connected through the planting plate; the top of the first aerosol box is set as an opening; the first spray system is arranged in the first aerosol box, and the second spray system is arranged in the second aerosol box; planting holes are arranged at intervals on the planting plate, and the annular rubber gasket is arranged above the planting plate and corresponds to the position of the planting holes up and down; the annular rubber gasket includes a stacked first gasket and a second gasket, the first gasket is arranged 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 gasket and the second gasket are provided with easy-tear openings.

3. The method for creating new potato germplasm with high resistance to late blight by 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 notch.

4. The method for creating new potato germplasm with high resistance to late blight by interspecific grafting according to claim 2, characterized in that: The nozzle of the second spray system extends downward by 10-20 cm from the junction of the bottom of the planting plate and the second aerosol culture box at an angle of 30-45 degrees for spraying.

5. The method for creating new potato germplasm with high resistance to late blight by interspecific grafting according to claim 1, characterized in that: In step (2), the grafting time is selected within one week before or after New Year's Day.

6. The method for creating new potato germplasm with high resistance to late blight by interspecific grafting according to claim 1, characterized in that: In step (4), the sodium chloride stress is to spray the leaves of the grafted seedlings 1 with a sodium chloride solution having a concentration of 100-150 mmol / L 1-2 times a day for 2-3 consecutive days, and spray until the leaves are wet each time; the inoculation of a small amount of Phytophthora refers to: using a spore concentration of 2×10 4 Spray one leaf of the grafted seedling with a suspension of Phytophthora spp. / mL, with a spraying amount of 1-2 mL per plant.

7. The method for creating new potato germplasm with high resistance to late blight by interspecific grafting according to claim 1, characterized in that: In S1 of step (4), the plant with a certain disease resistance refers to a plant with one of the following diseases: the plant has no disease spots on all leaves, some leaves have some disease spots, and less than 1 / 4 of the leaves of the plant have disease spots.

8. The method for creating new potato germplasm with high resistance to late blight by interspecific grafting according to claim 1, characterized in that: In step (4) S2, the method of inoculating a large amount of Phytophthora is as follows: using a spore concentration of 2×10 4 Spray 2 leaves of the grafted seedlings with a suspension of Phytophthora spp. / mL, with a spraying amount of 5-10 mL per plant.

9. The method for creating new potato germplasm with high resistance to late blight by interspecific grafting according to claim 1, characterized in that: In S2 of step (4), 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.

10. The method for creating new potato germplasm with high resistance to late blight by interspecific grafting according to claim 1, characterized in that: The mist cultivation device is placed in a temperature-controllable 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 mist cultivation box, the temperature control device comprises a temperature probe, a heating tube and a temperature controller, the temperature probe and the heating tube are electrically connected to the temperature controller respectively, and the temperature in the first mist cultivation box is controlled by the temperature controller; the light control device comprises an LED lamp and a time controller, the LED lamp and the time controller are connected, and the illumination time in the first mist cultivation box is controlled by the time controller; the humidity sensor is connected to the first spray system to control the air humidity in the first mist cultivation box.

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