A promoting healing liquid for grafted seedlings of melon vegetables by butt joint grafting

Abstract

The present invention discloses a promoting healing solution for grafted seedlings of cucurbit vegetables, which includes liquid A and liquid B. Liquid A contains paclobutrazol and potassium dihydrogen phosphate, and liquid B contains indole-3-acetic acid, kinetin, glucose and compound amino acids. The promoting healing solution for grafted seedlings can improve the matching of rootstock / scion of cucurbit vegetables, promote the connection of phloem and xylem of grafted cucurbit seedlings, achieve the rapid healing of grafted cucurbit seedlings under low night temperature conditions, shorten the healing time, and reduce the heating cost during the healing period.

Description

Technical Field

[0001] The present invention belongs to the technical field of vegetable seedling raising, and particularly relates to a promoting healing solution for grafted seedlings of cucurbit vegetables by butt joint grafting. Background Art

[0002] Cucurbit vegetables are rich in a large amount of vitamins, which have a certain regulating effect on human blood pressure and blood sugar, can maintain the sources of vitamins required for human health, and have high economic value and nutritional value, and are widely planted throughout China. Years of planting often face problems such as continuous cropping obstacles and soil-borne diseases. Grafting seedling raising is an important technical measure to overcome soil-borne diseases such as fusarium wilt, bacterial wilt, and verticillium wilt, enhance abiotic stresses such as drought tolerance and salt tolerance, promote the absorption of water and mineral nutrients, and improve yield, quality, and efficiency. More than 90% or even 100% of watermelon production in the main watermelon producing areas of Daxing District, Beijing, Anhui Province, Shandong Province, etc. in China adopts grafting cultivation.

[0003] The matching of the stem diameters of the rootstock and the scion is closely related to the success rate of butt joint grafting. Previous studies found that the stem diameters of melon scions of the same seedling age are all smaller than those of the rootstock. The rootstock variety with the smallest stem diameter - Weizhen, has a stem diameter of 2.228 mm 7 days after sowing, which is similar to the stem diameter of the scion 15 days after sowing. The rootstock variety with a larger stem diameter - Zhenzhuang, has a stem diameter of 3.057 mm 7 days after sowing, and the stem diameter of the scion only reaches 2.775 mm 23 days after sowing. The suitable grafting period for melons by butt joint grafting is the one-leaf and one-heart stage (10 - 15 days after sowing). If the seedling age is too large, it will cause the cotyledons to turn yellow and rot, and even lead to grafting failure; however, the melon seedlings at the one-leaf and one-heart stage have thin and weak stems, which are very different from the stem diameters of the rootstock, and are not conducive to grafting healing.

[0004] Grafting healing management is a key link in the production of grafted seedlings. During grafting, the vascular bundle tissues of the rootstock and scion are cut off, and the up and down transportation of substances is blocked. Environmental conditions of low light, high temperature, and high humidity can effectively regulate the formation of the isolation layer at the grafting healing site, the differentiation of callus, and the reconnection of vascular bundles, and promote the rapid and effective healing of grafted seedlings. Temperature is one of the decisive factors affecting grafting healing. The suitable grafting healing temperature for cucurbit vegetables is 23 - 28°C. However, the production of grafted seedlings in China is mainly carried out in the low-temperature season, and heating is required during the healing period. The higher the temperature, the higher the heating cost. Usually, the night temperature is controlled at a relatively low level (15 - 18°C). When the night temperature is 15°C, the healing period requires more than 7 days, and the seedling growth is slow. For the longer healing period, manual work such as uncovering the film for ventilation and covering the film for moisture preservation needs to be carried out every day. The management requirements are more stringent, which increases the labor cost, and the long healing period will increase the risk of rotten leaves, resulting in a decrease in survival rate and survival quality. Summary of the Invention

[0005] In order to improve the grafting survival rate of grafted seedlings of cucurbit vegetables, the present invention provides a promoting healing solution for grafted seedlings of cucurbits, which can increase the grafting survival rate of grafted seedlings of cucurbits by more than 95%. At the same time, it can achieve the rapid healing of grafted seedlings of cucurbits under low night temperature conditions, shorten the healing time, and reduce the heating cost during the healing period. In addition, this technology improves the matching degree of rootstock / scion and is suitable for mechanized operation.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A promoting healing solution for grafted seedlings of cucurbit vegetables, comprising solution A and solution B. Solution A contains paclobutrazol and KH2PO4, and solution B contains indole-3-acetic acid, kinetin, glucose and compound amino acids. Preferably, solution A contains 20 mg·L -1 paclobutrazol and 2 g·L -1 KH2PO4, and solution B contains 0.5 mg·L -1 indole-3-acetic acid, 10 mg·L -1 kinetin, 0.5% glucose and 0.1% compound amino acids.

[0008] Application of the above promoting healing solution in promoting the connection between the phloem and xylem of grafted seedlings of cucurbit vegetables.

[0009] A method for promoting the connection between the xylem and phloem of grafted seedlings of cucurbit vegetables: spraying solution A after the scion emerges from the soil, grafting 10 - 15 days after emerging from the soil, and spraying solution B on the grafted seedlings after grafting. Solution A contains 20 mg·L -1 paclobutrazol and 2 g·L -1 KH2PO4, and solution B contains 0.5 mg·L -1 indole-3-acetic acid, 10 mg·L -1 kinetin, 0.5% glucose and 0.1% compound amino acids.

[0010] The beneficial effects of the present invention are as follows: Solution A can thicken the scion stem, improve the matching degree of rootstock / scion, increase the grafting success rate, and is suitable for mechanized operation at the same time; Solution B can promote the connection between the phloem and xylem of grafted seedlings of cucurbits, and can achieve the rapid healing of grafted seedlings of cucurbit vegetables under low night temperature conditions, shorten the healing time, and reduce the heating cost during the healing period. Description of the Drawings

[0011] Figure 1 Shows the effects of exogenous auxin substances on the reconnection of phloem / xylem in grafted seedlings of melons. A, Phloem connection rate; B, Xylem connection rate.

[0012] Figure 2 Shows the effects of exogenous cytokinin on the reconnection of phloem / xylem in grafted seedlings of melons. A, Phloem connection rate; B, Xylem connection rate.

[0013] Figure 3 Effects of exogenous auxin and cytokinin on phloem / xylem reconnection in grafted seedlings. A, Phloem connection rate; B, Xylem connection rate.

[0014] Figure 4 Effects of exogenous sugar treatment on phloem / xylem of grafted seedlings. A, Phloem connection rate; B, Xylem connection rate.

[0015] Figure 5 Effects of exogenous hormones and sugars on phloem / xylem of grafted seedlings. A, Phloem connection rate; B, Xylem connection rate.

[0016] Figure 6 Variation of stem diameter of melon in Example 2.

[0017] Figure 7 Phloem connection of melon grafted seedlings in Example 3.

[0018] Figure 8 Xylem connection of melon grafted seedlings in Example 3. Detailed implementation manners

[0019] Example 1 Effects of exogenous substance treatment on melon grafting healing under low night temperature conditions

[0020] The tested melon scion variety was 'Minong', and the tested pumpkin rootstock variety was 'Deli No. 2'. The experiment was carried out in the Seedling Research Laboratory of the Institute of Crop Sciences, Wuhan Academy of Agricultural Sciences. The seeds were coated with Rovral and directly sown in plug trays filled with a mixed substrate of peat and perlite (3:1, v / v). The substrate humidity was 20%, and the sowing depth was 1.5 cm. After sowing, a layer of film was covered and placed in a germination chamber at 30°C for 48 - 72 h, and then removed when the seeds emerged. The scions were sown 4 days earlier than the rootstocks, and the rootstocks were grafted 10 days after sowing. The root-cutting approach grafting method was used for grafting. After grafting, different treatments were sprayed on the grafted plants. Each treatment had 3 replicates, and each replicate had 20 plants. CK was the control, treated with clear water. 10 days after grafting, the grafted plants were placed in an artificial climate chamber (28°C / 18°C day / night) for unified management.

[0021] The method for detecting phloem / xylem connection is as follows:

[0022] Preparation of 20 mg / mL Esculin working solution: Dissolve 0.4 g of Esculin in 20 mL of 60% acetonitrile solution (prepared with distilled water), and store it at room temperature in a brown reagent bottle. Preparation of 5 mg / mL Acid fushion working solution: Dissolve 0.5 g of AcidFuschin in 100 mL of distilled water, and store it at room temperature in a brown reagent bottle.

[0023] Phloem detection: Select the true leaves of the melon scion and sand the epidermis with sandpaper (without damaging the true leaves), drop 25 μl of 2.5 mM EDTA-2Na solution (to prevent the phloem from quickly closing), then drop 30 μl of Esculin working solution and spread it evenly, cover with a film, at a temperature of 28 °C, and light intensity of 50 μmol·m -2 ·s -1 . After 2 h, cut the stem segment (rootstock) 0.5 cm below the grafting interface, and observe and count the connectivity under a stereoscopic fluorescence microscope M205FA (Leica, Germany) with GFP. Phloem connectivity rate (%) = number of plants with fluorescence detected in the rootstock stem segment / total number of plants used for phloem detection × 100. Select 20 plants each time, continuously observe for 1 - 9 d after grafting, and repeat the experiment 3 times.

[0024] Xylem detection: Wash the roots of the grafted seedlings with water, only retain 1 cm long roots from the base of the stem, and keep the same root amount for each grafting combination; put the roots of the grafted seedlings into a centrifuge tube containing 1.5 ml of Acid fuchsin working solution, cover with a film, at a temperature of 28 °C, and light intensity of 50 μmol·m -2 ·s -1 . After 2 h, cut the stem segment (scion) 0.5 cm above the grafting interface, observe under a stereoscopic fluorescence microscope M205FA (Leica, Germany) with GFP, and xylem connectivity rate (%) = number of plants with fluorescence detected in the scion stem segment / total number of plants used for xylem detection × 100. Select 20 plants each time, continuously observe for 1 - 9 d after grafting, and repeat the experiment 3 times.

[0025] 1. Effects of exogenous auxin substances on the graft healing of melons

[0026] Treatments include: 50 mg / L IBA (IBA-50), 100 mg / L IBA (IBA-100), 300 mg / L IBA (IBA-300), 500 mg / L IBA (IBA-500); 0.5 mg / L IAA (IAA-0.5), 1 mg / L IAA (IAA-1), and 2 mg / L IAA (IAA-2).

[0027] Spraying 4 concentrations of IBA all had inhibitory effects on the graft healing of melons ( Figure 1 A and B). For example, at 6 d after grafting, compared with the CK treatment, the phloem connectivity rates of spraying 50 mg / L IBA, 100 mg / L IBA, 300 mg / L IBA, and 500 mg / L IBA decreased by 136.36%, 200.00%, 360.91%, and 457.14% respectively, and the xylem connectivity rates decreased by 40.00%, 70.93%, 181.44%, and 387.50% respectively.

[0028] Spraying IAA has a promoting effect on the xylem connection during the graft healing of melons ( Figure 1 B). As can be seen from Figure 1 B, on the 5th day after grafting, the xylem connection rates of spraying 0.5 mg / L IAA, 1 mg / L IAA, and 2 mg / L IAA increased by 236.00%, 89.00%, and 89.00% respectively compared with the CK treatment. There was no obvious difference in the phloem connection rate between the spraying IAA treatment and the CK treatment ( Figure 1 A).

[0029] 2. Effects of exogenous cytokinins on the graft healing of melons

[0030] The treatments included: 3 mg / L 6-BA (6-BA-3), 5 mg / L 6-BA (6-BA-5), 10 mg / L 6-BA (6-BA-10), 20 mg / L 6-BA (6-BA-20); 10 mg / L KT (KT10), 20 mg / L KT (KT20), and 30 mg / L KT (KT30).

[0031] Spraying 6-BA has no obvious promoting effect on the graft healing of melons, and shows an inhibitory trend with the increase of 6-BA concentration. For example, on the 6th day after grafting, spraying 3 mg / L 6-BA has no obvious effect on the graft healing. The phloem connection rates of spraying 5 mg / L 6-BA, 10 mg / L 6-BA, and 20 mg / L 6-BA decreased by 34.48%, 85.71%, and 254.55% respectively compared with the CK treatment ( Figure 2 A); the xylem connection rates decreased by 4.55%, 64.29%, and 187.50% respectively ( Figure 2 B).

[0032] Spraying KT has a promoting effect on the middle and late stages of the graft healing of melons, and the best effect is achieved at a concentration of 10 mg / L ( Figure 2 A and B). For example, on the 6th day after grafting, the phloem connection rate of spraying 10 mg / L KT increased by 11.54% compared with the CK treatment, and the phloem connection rates of spraying 20 mg / L KT and 30 mg / L KT decreased by 11.54% and 19.23% respectively compared with the CK treatment; the xylem connection rate of spraying 10 mg / L KT increased by 56.52% compared with the CK treatment, and there was no significant difference between spraying 20 mg / L KT and 20 mg / L KT compared with the CK treatment.

[0033] 3. Effects of the compounding of exogenous auxin and cytokinin on the graft healing of melons

[0034] The compounding of auxin and cytokinin in different ratios has different effects on the graft healing of melons. Spraying IBA50 and 6-BA in a ratio of 1:1 has an inhibitory effect on the graft healing of melons. For example, on the 5th day after grafting, the phloem connectivity rate of spraying IBA50 and 6-BA in a ratio of 1:1 decreased by 76.92% compared with the CK treatment; the xylem connectivity rate decreased by 37.00%( Figure 3 A).

[0035] When spraying IAA 1 and KT 20 in ratios of 1:1, 1:2, and 2:1, there is no obvious promoting effect on the phloem connectivity of the graft healing of melons, but there is a promoting effect on the xylem connectivity. For example, on the 5th day after grafting, the xylem connectivity rates of spraying IAA 1 and KT 20 in ratios of 1:1, 1:2, and 2:1 increased by 215.00%, 152.00%, and 131.00% respectively compared with the CK treatment( Figure 3 B).

[0036] When spraying IAA 0.5 and KT 10 in ratios of 1:1 and 2:1, there is a promoting effect on the phloem connection of the graft healing of melons. For example, on the 5th day after grafting, the phloem connectivity rate of spraying IAA 1 and KT 20 in a ratio of 1:1 increased by 42.99% compared with the CK treatment( Figure 3 A); on the 4th day after grafting, the phloem connectivity rate of spraying IAA 1 and KT 20 in a ratio of 2:1 increased by 72.06% compared with the CK treatment( Figure 3 A); while when spraying IAA 0.5 and KT 10 in ratios of 1:2, 1:3, and 3:1, there is no promoting effect on the phloem of the graft healing of melons( Figure 4 A). When spraying IAA0.5 and KT 10 in ratios of 1:1, 2:1, 1:2, 1:3, and 3:1, there is a promoting effect on the xylem connectivity of the graft healing of melons. For example, on the 5th day after grafting, the xylem connectivity rates increased by 246.50%, 257.00%, 83.75%, 119.56%, and 47% respectively compared with the CK treatment( Figure 3 B).

[0037] 4. Effects of exogenous sugars on the graft healing of melons

[0038] The treatments include: 0.5% glucose (P-0.5), 1% glucose (P-1), 2% glucose (P-2); 0.5% fructose (G-0.5), 1% fructose (G-1), 2% fructose (G-2), 0.5% sucrose (Z-0.5), 1% sucrose (Z-1), and 2% sucrose (Z-2).

[0039] Exogenous sugars of different types and concentrations have different effects on the graft healing of melons. Spraying sucrose at three concentrations did not promote the graft healing of melons and showed an inhibitory effect in the early stage of graft healing. For example, at 4 days after grafting, the phloem connection rates of spraying 0.5% sucrose, 1% sucrose, and 2% sucrose were decreased by 466.67%, 277.78%, and 277.78% respectively compared with the CK treatment( Figure 4 A); there was no obvious difference in the xylem connection rate( Figure 4 B).

[0040] Spraying fructose at three concentrations promoted the phloem connection of the graft healing of melons. For example, at 5 days after grafting, the phloem connection rates of spraying 0.5% fructose, 1% fructose, and 2% fructose were increased by 26.92%, 38.46%, and 44.23% respectively compared with the CK treatment( Figure 4 A), while there was no obvious difference in the xylem connection rate( Figure 4 B).

[0041] Spraying glucose at three concentrations promoted the graft healing of melons. For example, at 4 days after grafting, the phloem connection rates of spraying 0.5% glucose, 1% glucose, and 2% glucose were increased by 94.12%, 94.12%, and 58.82% respectively compared with the CK treatment( Figure 4 A); in terms of the xylem connection rate, spraying 0.5% glucose promoted the xylem connection in the early stage of the graft healing of melons. At 4 days after grafting, the xylem connection rate of spraying 0.5% glucose was increased by 117.24% compared with the CK treatment, and there was no significant difference between spraying 1% glucose and 2% glucose compared with the CK treatment( Figure 4 B).

[0042] 5. Effects of the compounding of exogenous hormones and sugars on the graft healing of melons

[0043] The compounding of exogenous hormones and sugars in different ratios has different effects on the graft healing of melons. Spraying IAA 0.5, KT 10, and P 0.5 in a ratio of 1:1:1 had a significant promoting effect on the phloem connection of the graft healing of melons. For example, at 5 days after grafting, the phloem connection rate of spraying IAA 0.5, KT 10, and P 0.5 in a ratio of 1:1:1 was increased by 55.77% compared with the CK treatment( Figure 5 A).

[0044] Spraying IAA 0.5, KT 10 and P 0.5 in the ratios of 1:1:1, 2:2:1 and 1:1:2 had a significant promoting effect on the xylem connection during the graft healing of melons. For example, on the 5th day after grafting, the xylem connection rates of spraying IAA 0.5, KT 10 and P 0.5 in the ratios of 1:1:1, 2:2:1 and 1:1:2 increased by 173.00%, 131.00% and 89.00% respectively compared with the CK treatment ( Figure 5 B).

[0045] In summary, spraying 0.5 mg / L IAA or 10 mg / L KT can promote the xylem connection rate during the grafting of melons. Spraying 0.5% glucose can promote the phloem connection rate during the grafting of melons. Spraying IAA 0.5 mg / L and KT 10 mg / L in the ratio of 1:1 has a significant promoting effect on the connection of both the phloem and xylem during the graft healing of melons. Spraying IAA 0.5 mg / L, KT 10 mg / L and glucose 0.5% in the ratio of 1:1:1 has a significant promoting effect on the connection of both the phloem and xylem during the graft healing of melons, and the promoting effect is the best.

[0046] Example 2 Effects of the compound application of paclobutrazol, potassium dihydrogen phosphate and calcium agent on the stem diameter of melon scions

[0047] The treatments included water (CK), 30 mg·L -1 paclobutrazol (C1), 30 mg·L -1 paclobutrazol + 3 g·L -1 KH2PO4 (C2), 30 mg·L -1 paclobutrazol + 3 g·L -1 KH2PO4 + 3 g·L -1 chelated calcium CaM (C3) and 30 mg·L -1 paclobutrazol + 3 g·L -1 chelated calcium CaM (C4). A total of 5 treatments were set, with 3 replicates for each treatment. The tested melon scion variety was 'Minong'. After the scions emerged from the soil, 25 mL of the treatment solution was sprayed on each tray by foliar spraying, and the stem diameter of the plug seedlings was measured at fixed plants.

[0048] The compound application of paclobutrazol, potassium dihydrogen phosphate and calcium agent can increase the stem diameter of melon scions ( Figure 6 ). In the early stage of treatment (7 days and 10 days after treatment), there was no significant difference in the stem diameter of the watermelon scions in each treatment compared with the control. In the middle stage of treatment (13 days, 16 days and 19 days after treatment), the C2 treatment (30 mg·L -1 paclobutrazol + 3 g·L -1The stem diameters of watermelon scions treated with KH2PO4 were significantly increased by 8.02%, 8.12% and 12.29% respectively compared with the control, and there were no significant differences between the other treatments and the control; at the later stage of treatment (22 days after treatment), the stem diameters of watermelon scions treated with C1, C2 and C3 were significantly increased compared with the control, by 8.33%, 13.35% and 10.13% respectively.

[0049] Example 3 Healing test of melon grafted seedlings under the temperature condition of 28°C / 18°C

[0050] The tested melon scion variety was 'Minong', and the tested pumpkin rootstock variety was 'Deli No. 2'. The seeds were directly sown in plug trays filled with a mixed substrate of peat and perlite (3:1, v / v) after being coated with Rydon. The substrate humidity was 20%, and the sowing depth was 1.5 cm. After sowing, a layer of film was covered and placed in a germination chamber at 30°C for germination for 48 - 72 h, and then removed when the seeds emerged. The scions were sown 4 days earlier than the rootstocks. Five days after sowing the rootstocks, after the scion seedlings emerged, the grafted seedling healing-promoting liquid A (20 mg·L -1 paclobutrazol + 2 g·L - 1 KH2PO4) was sprayed, 25 mL per tray. Grafting was carried out 15 days after sowing the rootstocks, using the root-cutting butt joint method for grafting. After grafting, the grafted seedling healing-promoting liquid B (0.5 mg·L -1 IAA + 10 mg·L -1 KT + 0.5% glucose + 0.1% compound amino acids) was sprayed, 25 mL per tray, placed on a healing shelf and covered with a film to keep moisture. The temperature of the healing chamber was set at 28°C / 18°C (day / night). The connection of the phloem and xylem of melon grafted seedlings during the grafting healing period was measured, and the method was referred to Example 1. CK was the control, treated with clear water.

[0051] It was found that the grafted seedling healing-promoting liquid could promote the connection of the phloem and xylem of melon grafted seedlings. Through the treatment with the healing-promoting liquid, the phloem was connected 3 days after grafting and reached 90% 5 days after grafting; for the untreated grafted seedlings, the phloem was connected 4 days after grafting and reached 86.67% 6 days after grafting ( Figure 7 ). The connection of the phloem of melon grafted seedlings treated with the grafted seedling healing-promoting liquid was advanced. On the 4th day after grafting, the xylem connection rate of the seedlings treated with the healing-promoting liquid reached 23.33%, while that of the untreated grafted seedlings was only 9.21%; on the 7th day after grafting, the xylem connection rate of the seedlings treated with the healing-promoting liquid reached 100%, while that of the untreated grafted seedlings was only 75.56% ( Figure 8 ).

Claims

1. A promoting healing liquid for grafted seedlings of cucurbit vegetables, characterized in that, It consists of liquid A and liquid B. Liquid A consists of 20 mg∙L -1 paclobutrazol and 2 g∙L -1 potassium dihydrogen phosphate. Liquid B consists of 0.5 mg∙L -1 indole-3-acetic acid, 10 mg∙L -1 kinetin, 0.5% glucose and 0.1% compound amino acids.

2. Use of the promoting healing liquid according to claim 1 in promoting the connection of phloem and xylem of grafted seedlings of cucurbitaceous vegetables by sticking and grafting.

3. A method for promoting the connection of the xylem and phloem of grafted melon vegetables, characterized in that, Spray solution A after the scion emerges from the soil, and graft 10 - 15 days after the scion emerges from the soil. The solution A consists of 20 mg∙L -1 paclobutrazol and 2 g∙L -1 KH2PO4; spray solution B on the grafted seedlings after grafting. The solution B consists of 0.5 mg∙L -1 indole-3-acetic acid, 10 mg∙L -1 kinetin, 0.5% glucose and 0.1% compound amino acids.

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