Application of TOR inhibitor in growth and development of cucumber grafted seedlings

Abstract

The invention relates to an application of a TOR inhibitor in growth and development of a cucumber grafted seedling, and a use method of the TOR inhibitor in the cucumber grafted seedling comprises the following steps: uniformly spraying the TOR inhibitor on a scion cotyledon of the grafted seedling for the 0th day, the 1st day, the 3rd day and the 5th day after the grafted seedling is grafted; the dosage of the TOR inhibitor is 10 to 100 M. According to the application, cucumber 'R1461' is taken as a scion, pumpkin 'Zhongshu rootstock 1' is taken as a rootstock, grafting is performed by adopting a sleeve grafting method, a CsTOR inhibitor AZD8055 is sprayed from an external source, and the concentration for inhibiting healing of a cucumber-pumpkin grafting interface is screened out; after 0M, 10M, 25M, 50M and 100M TOR inhibitors (AZD8055) are sprayed on cucumber-pumpkin grafted seedlings respectively, survival rate and physiological index determination and vascular bundle reconnection observation are carried out, and it is found that the exogenous TOR inhibitor AZD8055 has a concentration effect on grafting interface healing.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural planting, and in particular to application of a TOR inhibitor in the growth and development of grafted cucumber seedlings. Background Art

[0002] Cucumber (Cucumis sativus L.), also known as cucumber and green melon, is one of the important greenhouse vegetable crops in my country. In the cultivation and production of cucumbers, it is often affected by biological and abiotic stresses. Grafting technology has the advantages of improving cucumber yield and fruit quality, preventing continuous cropping obstacles, resisting soil-borne diseases and stress resistance, and has been widely used in greenhouse cucumber production. Therefore, the development of the grafted cucumber industry is conducive to driving local economic development, promoting farmers' income, and promoting the modernization of agriculture and rural areas.

[0003] Grafting is a technique that purposefully grafts the buds or branches of one plant to the appropriate position of another plant with a root system, so that the two can be combined into a new plant. Among them, the healing of the graft interface directly determines the graft affinity. At present, in the production of grafted cucumber seedlings in my country, the slow healing speed of the graft interface and the instability of healing have resulted in a low degree of mechanization and high management costs in the production of grafted seedlings, which is also the main factor restricting the development of my country's cucumber grafted seedling industry. Vascular bundle reconnection is the key to graft healing, and its regulatory mechanism is still unclear. Cucumber, as a Cucurbitaceae crop with double-tough vascular bundles, is one of the model plants for vascular bundle research, providing material advantages for the study of graft healing. Therefore, the study of the mechanism of vascular bundle reconnection in cucumber-pumpkin graft healing not only has an important guiding role in the intensive production of cucumber grafted seedlings and the development and utilization of high-quality cucumber rootstock germplasm, but also has important scientific significance for in-depth analysis of the molecular mechanism of heterologous graft healing. At present, there are many developments on grafting technology for horticultural melon crops, but most of them are concentrated on grafting methods and grafting rootstock breeding, while technical research on promoting the grafting healing process has not been reported. Shortening the grafting healing time can effectively reduce the cost of grafted seedlings and ensure a stable survival rate of grafted seedlings. Therefore, research on technology to promote grafting healing of melon crops is urgent.

[0004] Target of Rapamycin TOR (Target of Rapamycin) is a highly conserved serine / threonine protein kinase in eukaryotes, playing a core role in the growth and metabolism of eukaryotes (such as yeast, algae, animals and plants). Studies in plants have found that TOR is a central regulator of plant growth and development. In most plants, there is only one TOR Gene, TORIn plants, TOR can not only integrate hormones, external environment and other signals to coordinate processes such as cell growth and cell division, but also affect the entire life cycle of plants from embryonic development to aging and death. Studies have found that the phosphorylation substrates of TOR kinase are mostly plant hormone-related proteins, including transcription factors E2Fa / b, auxin export protein PIN2, ethylene signal protein EIN2 and ABA receptor PYLs. Sugar activates TOR activity and stabilizes E2Fa protein, coupling auxin signal ARF-LBD to promote tissue dedifferentiation to form callus and cell proliferation. ABA-responsive gene YAK1 is also a downstream target gene of TOR, which mainly regulates meristem activity and differentiation to regulate root growth, including protoxylem and vascular tissue. In addition, when the tobacco mutant of TOR downstream inhibitory signal atg5 was grafted into Arabidopsis, the graft survival rate was significantly reduced, indicating that TOR-induced cell autophagy is essential for graft interface healing. In recent years, many breakthroughs have been made in the study of plant TOR in cell proliferation, tissue differentiation and growth, and its conservative biological functions and plant-specific signal pathways have been discovered, but its function in graft healing has rarely been reported.

[0005] In animals and yeast, basic research on the TOR pathway has benefited from the inhibitor rapamycin, a target of TOR, and the ATP competitive inhibitor asTORis, including PP242, Torin1, KU-0063794, WYE-354, Torin2, AZD-8055, WYE-125132, etc. Because rapamycin does not inhibit all TORC1 activities and is likely not to inhibit the activity of potential new TOR complexes in plants, asTORs are widely used in plants. In terms of inhibitory effect, AZD-8055>KU-0063794, WYE-132>WYE-354; the above asTORs inhibitors have become effective exogenous substances for studying TOR function in plants. Among them, Torin1 is less used because of its difficulty in solubility, while AZD-8055 and WYE-132 have good solubility, repeatability and inhibitory effect, and have concentration effects, and are widely used in plants. In addition, although AZD-8055 and WYE-132 can effectively inhibit the activity of plant TOR complex and thus inhibit plant growth, considering the cost issue, more studies choose AZD-8055 as a TOR inhibitor. Whether TOR inhibitors are effective in graft interface healing needs further in-depth research. Summary of the invention

[0006] The present invention aims to solve the problem of low survival rate of grafted cucumber seedlings and provides the following technical solutions: In a first aspect, the present invention provides an application of a TOR inhibitor in the growth and development of grafted cucumber seedlings. The method for using the TOR inhibitor in grafted cucumber seedlings is as follows: spraying the TOR inhibitor evenly on the cotyledons of the scion of the grafted seedlings, and the spraying time is respectively 0, 1, 3, and 5 days after the grafting of the grafted seedlings; the dosage of the TOR inhibitor is 10-100 µM.

[0007] Furthermore, the cucumber grafted seedlings use 11-day-old cucumber seedlings as scion and 7-day-old pumpkin seedlings as rootstock. A sharp new blade is used to make a 30° oblique cut from the rootstock hypocotyl in the vertical direction of the cotyledons, and the rootstock above the incision is completely removed (including the cotyledons, growth point, and part of the hypocotyl). Then, a blade is used to make a 30° oblique cut along the cotyledon direction 2-2.5 cm below the cotyledons of the scion. After cutting, the two are tightly pressed together and fixed with a grafting clamp to form a grafted seedling.

[0008] Furthermore, the TOR inhibitor is specifically AZD8055.

[0009] Furthermore, when the dosage of the TOR inhibitor is 10 μM, the survival rate of cucumber grafted seedlings can be improved, the reconnection of the vascular bundles at the grafting interface of the cucumber grafted seedlings can be promoted, and the leaf area and hypocotyl thickness of the grafted seedlings can be increased to promote the growth of the grafted seedlings.

[0010] Based on the above technical solution, when the dosage of the TOR inhibitor is 100 µM, the survival rate of cucumber grafted seedlings can be reduced, the reconnection of vascular bundles at the interface of cucumber grafted seedlings can be inhibited, and the growth of grafted seedlings can be inhibited.

[0011] Further preferably, when the dosage of the TOR inhibitor is 100 µM to inhibit the growth of grafted seedlings, 5 µM NAA is sprayed exogenously to alleviate the effect of 100 µM TOR inhibitor on the growth and development of grafted cucumber seedlings.

[0012] In a second aspect, the present invention further provides a growth promoter for grafted cucumber seedlings, wherein the promoter comprises the TOR inhibitor AZD8055 as described in claim 3.

[0013] The present invention has the following advantages / benefits: cucumber( Cucumis sativusL.) is one of the important greenhouse vegetable crops in my country. Cucumber grafted seedlings are often used for production in greenhouse cultivation. Target of Rapamycin protein TOR (Target of Rapamycin) is a central regulator of plant growth and development, but it is rarely reported in the healing of grafted interfaces. In this study, cucumber 'R1461' was used as the scion and pumpkin 'Zhongshuzhen No. 1' was used as the rootstock. The grafting was carried out by sleeve grafting. The concentration that inhibited the healing of cucumber-pumpkin grafted interface was screened by exogenous spraying of CsTOR inhibitor AZD8055. The survival rate, physiological indexes and vascular bundle reconnection were measured after the cucumber-pumpkin grafted seedlings were sprayed with 0 µM, 10 µM, 25 µM, 50 µM and 100 µM TOR inhibitor (AZD8055) respectively. The results showed that the exogenous TOR inhibitor AZD8055 had a concentration effect on the healing of grafted interfaces. Treatment with 10 µM TOR inhibitor can improve the survival rate of cucumber-squash grafted seedlings, promote the reconnection of vascular bundles at the graft interface, and thus promote the growth of grafted seedlings; while treatment with high concentration of 100 µM TOR inhibitor has the opposite effect, mainly inhibiting the reconnection process of xylem at the graft interface, and through concentration screening experiments, it was found that 100 µM is the appropriate TOR inhibitor concentration. And after re-application of auxin to cucumber-squash grafted seedlings treated with 100 μM TOR inhibitor, exogenous auxin can effectively alleviate the inhibitory effect of high concentration TOR inhibitor on the reconnection of vascular bundles at the graft interface of cucumber-squash grafted seedlings and the growth of grafted seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0015] Figure 1 : Effects of different concentrations of TOR inhibitors of the present invention on vascular reconnection of cucumber-pumpkin grafted seedlings; (A) phloem reconnection statistics, (B) xylem reconnection statistics; Figure 2 : Effects of different concentrations of TOR inhibitors of the present invention on cucumber-pumpkin grafting survival rate (A), grafted seedling phenotype (B) and various phenotypic indicators (CF); Figure 3 : Effects of NAA and TOR inhibitors of the present invention on vascular reconnection of cucumber-pumpkin grafted seedlings after treatment; (A) phloem reconnection statistics, (B) xylem reconnection statistics; Figure 4: Effects of NAA and TOR inhibitor treatments of the present invention on the survival rate (A), grafted seedling phenotype (B) and various phenotypic indicators (CF) of cucumber-squash grafting. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0017] Embodiment 1: 1.1 Materials and methods 1.1.1 Experimental materials and growth conditions Cucumber variety 'R1461' and pumpkin variety 'Zhongshuzhen No. 1' were used to construct the cucumber-pumpkin grafting combination. 'R1461' cucumber germplasm was provided by Zhejiang Academy of Agricultural Sciences, and 'Zhongshuzhen No. 1' pumpkin was purchased from Zhongshu Seed Technology (Beijing) Co., Ltd.

[0018] AZD8055 was purchased from Adooq Company with the CAS number of 1009298-09-2.

[0019] 1.1.2 Grafting methods and management methods Plant seedling raising and management were carried out in the climate chamber of the College of Horticultural Science, Zhejiang A&F University. Select complete and full cucumber seeds, wrap them with clean gauze, and soak them in warm water at 55 ℃ for 2-3 hours. Then transfer the seeds to wet filter paper and germinate them in a petri dish at 28 ℃ in the dark for 12-24 hours. Pumpkin seeds are germinated 2 days later than cucumber seeds. Soak the seeds at the same temperature for 5-6 hours and germinate them at 28 ℃ in the dark for 24-36 hours. When most of the seeds are white, sow them in a 50-hole tray filled with substrate (peat: vermiculite = 2:1) and place them in an artificial climate chamber for cultivation.

[0020] Culture conditions: 12 h light / 12 h dark, light intensity of 50,000 Lux, day temperature of 28±1°C / night temperature of 18±1°C, relative air humidity maintained at 65–85%.

[0021] The grafting was performed by sleeve grafting, and the detailed process is described in Miao et al. (2019b). Using 11-day-old cucumber seedlings as scions and 7-day-old pumpkin seedlings as rootstocks, a sharp new blade was used to make a 30° oblique cut from the rootstock hypocotyl along the vertical direction of the cotyledons, and the entire rootstock above the incision was removed (including cotyledons, growth points, and part of the hypocotyl). Then, a blade was used to make a 30° oblique cut along the cotyledon direction 2-2.5 cm below the cotyledons of the scion. After cutting, the two were placed close together and fixed with a grafting clamp.

[0022] After grafting, a transparent plastic tray cover was used to seal the seedlings to maintain high humidity to prevent the grafted seedlings from being damaged by wind in the early stage. At the same time, the sealed tray was moved into the artificial climate room for management measures such as heat preservation and moisture retention, light conditions, and wind protection. This day was defined as 0 Days After Grafting (0 DAG). The specific measures for grafting management are as follows: Light management: From 0 to 3 DAG, the grafted seedlings were kept in the dark; from 3 to 5 DAG, they were managed under weak light (5000 Lux); from 6 to 7 DAG, they were gradually put into normal light (8000 Lux) conditions. Ventilation management: The humidity was kept above 95% in the first 4 days (0-3 DAG), and the vents of the transparent plastic tray cover were opened for 2-3 h from the 5th to the 8th day (4-7 DAG), and the ventilation time was gradually increased to gradually reduce the humidity to about 75%. After 7 DAG, the grafted seedlings survived and were managed normally.

[0023] 1.1.3 Treatment with TOR inhibitor AZD8055, auxin and 6-BA Cucumber-squash grafted seedlings were uniformly sprayed with different concentrations of TOR inhibitor AZD8055 (0, 10, 25, 50 and 100 µM) at the same time. The spraying site was the cotyledons of the scion of the grafted seedlings, and the spraying time was 0, 1, 3, and 5 days after the grafting of the cucumber-squash grafted seedlings, with 50-100 plants in each treatment.

[0024] The spraying method of auxin NAA (5 µM) was the same as that of TOR inhibitor AZD-8055.

[0025] 1.1.4 Phenotypic index measurement and survival rate statistics of grafted plants At 0 DAG, the initial root length of the rootstock was measured. At 8 DAG, 10 grafted seedlings were randomly selected from each treatment to measure physiological parameters, including fresh weight, dry weight, area of ​​the first true leaf of the scion, hypocotyl thickness of the scion and the rootstock, and root length of the rootstock. Before weighing the fresh weight of the grafted seedlings, the roots of the grafted seedlings must be cleaned, wiped dry with paper, and weighed and recorded using an electronic balance. The plant was placed in a paper bag of appropriate size and dried in an oven at 105 °C for 15 min, then dried in an oven at 70 °C for 2 days, and the dry weight of the cucumber-pumpkin grafted seedlings was weighed using a micrometer balance. The measurement and calculation method of the first true leaf area was calculated using the correction coefficient K=0.743 obtained by Pei Xiaobo et al. (2005), and the formula was S=KL (S: first true leaf area; K: correction coefficient; L: first true leaf length × first true leaf width). The hypocotyl thickness of the scion and stock and the root length of the stock were measured using a vernier caliper and a ruler, respectively.

[0026] The survival rate of cucumber-pumpkin grafted seedlings was calculated on DAG 8. Survival rate = (number of grafted seedlings with good growth in each treatment ÷ total number of grafted seedlings in each treatment) × 100%.

[0027] 1.1.5 Analysis of phloem and xylem reconnection The reconnection rates of phloem and xylem were measured by the following methods. 5(6)-CFDA (7C0503, biotium) was used for phloem reconnection observation, and acid fuchsin (RH338273, rhawn) was used for xylem reconnection observation. The reconnection rate was expressed as (number of grafted seedlings observed to be reconnected / total number of observations) × 100%. Cucumber / squash grafted seedlings were randomly sampled at 0, 1, 3, 5, and 7 days after grafting, and 15-20 plants were randomly sampled each time. The method for observing phloem reconnection was as follows: First, a blade was used to gently lift the epidermis of about 1-2 mm long near the growth point of the scion cotyledon to create a slight wound, and 2 µL of 1 mM CFDA was placed on the wound. After 3 hours, a cross-section of the scion stock 0.5 cm above and below the grafting interface was taken to observe the fluorescence (fluorescence type: GFP) transport status under a fluorescence microscope (OLYMPUS DP80). Observation method of xylem reconnection: The roots of the grafted seedlings must be carefully cleaned with water, wiped dry, and placed in 0.1% (w / v) fuchsin dye solution, with the roots just completely immersed. Place at room temperature for 1.5 h, and take cross-sections of the scion and stock 0.5 cm above and below the grafting interface to observe the fluorescence (fluorescence type: TXRED) transport status under a fluorescence microscope (OLYMPUS DP80).

[0028] Example 2: Effects of different concentrations of TOR inhibitor AZD8055 on the healing of grafted interfaces of cucumber-pumpkin grafted seedlings In order to clarify the effect of TOR inhibitors on the healing of grafted seedling grafting interfaces, the reconnection status of phloem and xylem was determined by dyeing with CFDA and acid fuchsin fluidity, respectively. Figure 1 The results showed that TOR inhibitors significantly affected the healing of vascular bundle reconnection at the graft interface of cucumber-pumpkin grafted seedlings, and the effect on xylem reconnection was greater than that on phloem reconnection.

[0029] Compared with the control, the phloem and xylem reconnection of the grafted seedlings treated with 10 µM AZD8055 increased significantly from 3 DAG (Day After Grafting), while there was no significant difference in phloem reconnection of the grafted seedlings treated with 100 µM AZD8055, but the xylem reconnection was significantly lower than that of the control group (CK) from 5 DAG. The vascular reconnection of the grafted seedlings treated with 25 µM and 50 µM AZD8055 was not significantly different from that of the control group (CK). Figure 1 , AB). Therefore, it was concluded that 10 µM AZD8055 could promote the healing of the vascular bundle at the grafting interface of grafted seedlings, including the reconnection of phloem and xylem, while 100 µM AZD8055 mainly affected the healing of the xylem at the interface, thereby delaying the reconnection of the vascular bundle, which was one of the reasons why it significantly inhibited the growth of grafted seedlings.

[0030] Figure 1 and Figure 2 In the table, CK refers to treatment without AZD8055; 10A refers to treatment with 10 µM AZD8055; 25A refers to treatment with 25 µM AZD8055; 50A refers to treatment with 50 µM AZD8055; and 100A refers to treatment with 100 µM AZD8055.

[0031] Example 3: Effects of different concentrations of TOR inhibitor AZD8055 on the growth of cucumber-squash grafted seedlings To clarify the effect of TOR inhibitor AZD8055 on the growth of cucumber-squash grafted seedlings, cucumber-squash grafted seedlings were treated with AZD8055 at 10 µM, 25 µM, 50 µM and 100 µM, and 0 µM AZD8055 was used as the control group. The experimental results are shown in Figure 2 shown.

[0032] The survival rate of the grafted seedlings was measured on the 8th day after grafting ( Figure 2 -A) Statistics show that with the increase of TOR inhibitor concentration, the graft survival rate shows a trend of first increasing and then decreasing. The survival rate of cucumber-squash grafted seedlings treated with 10 µM AZD8055 was significantly higher than that of the control group (CK), reaching 80%. The survival rates of grafted seedlings treated with 25 µM, 50 µM and 100 µM AZD8055 were significantly lower than those of the control group (CK), at 52.52%, 49% and 37%, respectively. The above conclusions show that cucumber-squash grafted seedlings have a concentration effect on TOR inhibitors.

[0033] The phenotypes of cucumber-squash grafted seedlings treated with different concentrations of TOR inhibitors were observed. It was found that the growth and development trend of cucumber-squash grafted seedlings treated with 10 µM ZD8055 was faster than that of the control group (CK), while the growth of grafted seedlings treated with 50 µM and 100 µM ZD8055 was obviously inhibited, and the true leaves of the grafted seedlings were wrinkled and the roots were shortened ( Figure 2 -B).

[0034] The fresh and dry weight of grafted seedlings during the graft healing period was measured ( Figure 2 -C), leaf area ( Figure 2 -D), hypocotyl thick ( Figure 2 -E), root length ( Figure 2 -F) and other indicators. With the increase of AZD8055 treatment concentration, most of the measured indicators showed a trend of first increasing and then decreasing. Compared with the control group, the dry and fresh weights of the grafted seedlings were significantly reduced after 100 µM AZD8055 treatment. After 10 µM and 50 µM AZD8055 treatment, only a single indicator (dry weight or fresh weight) of the grafted seedlings showed significant changes, while there was no significant difference in 25 µM AZD8055 treatment ( Figure 2 -C). It can be seen that 100 µM AZD8055 can significantly inhibit the growth of cucumber-squash grafted seedlings.

[0035] The leaf area and hypocotyl thickness of the grafted seedlings and scion and rootstock treated with 10 µM AZD8055 were significantly higher than those of the control group (CK), increasing by 3.26 cm 2 , 0.16 mm, 0.09 mm, while the 100 A treatment significantly reduced the leaf area of ​​the grafted seedlings and the hypocotyl thickness of the scion ( Figure 2 , DE). In the root length statistics, we found that the root system of the grafted seedlings treated with AZD8055 was significantly shorter than that of the control group (CK), especially the grafted seedlings treated with 100 µM AZD8055, which was shortened by about 50% ( Figure 2 -F). In summary, TOR inhibitors affect the growth of cucumber-squash grafted seedlings in a concentration-dependent manner.

[0036] Example 4: Effect of exogenous NAA spraying on vascular bundle reconnection at the cucumber-squash interface after treatment with 100 µM TOR inhibitor In order to further clarify whether auxin would alleviate the effect of TOR inhibitors on the healing of the grafted interface of cucumber-squash grafted seedlings, CFDA and acid fuchsin reagents were used again to stain the phloem and xylem, respectively. The results showed that spraying auxin could alleviate the effect of high concentrations of inhibitors on the healing of grafted seedlings.

[0037] In the observation of phloem reconnection, it was found that the spraying of auxin and TOR inhibitor alone or in combination had no significant difference compared with the control group, but had a very significant effect on xylem reconnection. Among them, the 100 µM TOR inhibitor treatment at 4 DAG and then the 5 µM NAA treatment showed a significant difference from the 100 µM TOR inhibitor treatment alone, but there was no significant difference from the control group, and this phenomenon continued until 7 DAG ( Figure 3 -A, B). This indicates that spraying 5 µM NAA after 100 µM TOR inhibitor treatment alleviated the effect of high concentration inhibitor on the healing and reconnection of the grafted interface of grafted seedlings on the 4th day after grafting.

[0038] Figure 3 and Figure 4 In the data, CA refers to treatment without AZD8055; 100A refers to treatment with 10 µM AZD8055 alone; 5N refers to treatment with 5 µM NAA alone; 100A+5N (100A / 5N) refers to treatment with 100 µM AZD8055 followed by 5 µM NAA.

[0039] Example 5: Effect of exogenous NAA spraying on the growth and development of cucumber-squash grafted seedlings after treatment with 100 μM TOR inhibitor In order to clarify whether auxin can alleviate the effect of 100 µM TOR inhibitor AZD8055 on the growth and development of cucumber-squash grafted seedlings, 5 µM NAA treatment was added while spraying 100 µM TOR inhibitor to form 4 combinations (CK, 100A, 5N, 100A / 5N). The survival rate of grafted seedlings on the 8th day after grafting was statistically analyzed and it was found that the survival rates of CK, 100A, 5N, and 100A / 5N were 63.65%, 27.24%, 75.73%, and 50.45%, respectively. Spraying auxin alone can effectively promote the survival rate of cucumber-squash grafted seedlings, and the spraying of auxin significantly increased the survival rate of cucumber-squash grafted seedlings after TOR was inhibited ( Figure 4 -A).

[0040] The phenotypes of cucumber-squash grafted seedlings treated with the four combinations were observed. It was found that the growth and development trend of the cucumber-squash grafted seedlings treated with 5 µM NAA was better than that of the control group (CK). The growth of the grafted seedlings treated with 100 µM AZD8055 was still severely inhibited. However, the growth trend of the cucumber-squash grafted seedlings treated with 100 µM TOR inhibitor AZD8055 was restored by spraying auxin, and the inhibition of leaves and roots was almost alleviated ( Figure 4 -B).

[0041] Based on the above phenomenon, more detailed phenotypic indicators (dry and fresh weight, leaf area, hypocotyl thickness, root length, etc.) were measured for grafted seedlings, and it was found that the increase of auxin can alleviate the effect of TOR inhibitors on the growth and development of cucumber-squash grafted seedlings. In all physiological indicators, the grafted seedlings treated with 100 µM AZD8055 were always lower than the control group (CK), while the growth of spraying alone was the opposite ( Figure 4 -C~F). When auxin and TOR inhibitor were used to treat cucumber-pumpkin grafted seedlings at the same time, it was found that the dry and fresh weight of cucumber-pumpkin grafted seedlings affected by auxin was significantly higher than that of the control group (CK), and the leaf area, hypocotyl thickness of scion and rootstock, and root length were also restored ( Figure 4 -C~F). This proves that auxin can effectively alleviate the effect of TOR inhibitors on the growth of cucumber-squash grafted seedlings.

[0042] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. The application of TOR inhibitor in the growth and development of cucumber grafted seedlings, characterized in that: The method for using the TOR inhibitor in cucumber grafted seedlings is: spraying the TOR inhibitor evenly on the scion cotyledons of the grafted seedlings, and the spraying time is respectively 0, 1, 3, and 5 days after the grafted seedlings are grafted; the dosage of the TOR inhibitor is 10-100 μM.

2. The use of the TOR inhibitor according to claim 1 in the growth and development of cucumber grafted seedlings, characterized in that: The cucumber grafted seedlings are grafted by using 11-day-old cucumber seedlings as scions and 7-day-old pumpkin seedlings as rootstocks.

3. The use of the TOR inhibitor according to claim 1 in the growth and development of cucumber grafted seedlings, characterized in that: The TOR inhibitor is specifically AZD8055.

4. The use of the TOR inhibitor in the growth and development of cucumber grafted seedlings according to claim 1, characterized in that: When the dosage of the TOR inhibitor is 10 μM, the survival rate of cucumber grafted seedlings can be improved, the reconnection of the vascular bundles at the grafting interface of the cucumber grafted seedlings can be promoted, and the leaf area and hypocotyl thickness of the grafted seedlings can be increased to promote the growth of the grafted seedlings.

5. The use of the TOR inhibitor in the growth and development of cucumber grafted seedlings according to claim 1, characterized in that: When the dosage of the TOR inhibitor is 100 μM, the survival rate of cucumber grafted seedlings can be reduced, the reconnection of the vascular bundles at the interface of the cucumber grafted seedlings can be inhibited, and the growth of the grafted seedlings can be inhibited.

6. The use of the TOR inhibitor in the growth and development of cucumber grafted seedlings according to claim 5, characterized in that: When the dosage of the TOR inhibitor is 100 µM to inhibit the growth of the grafted seedlings, 5 µM NAA is sprayed exogenously to alleviate the effect of the 100 µM TOR inhibitor on the growth and development of the grafted cucumber seedlings.

7. A cucumber grafted seedling growth promoter, characterized in that: The promoter comprises the TOR inhibitor AZD8055 according to claim 3.

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