Method for promoting flowering dogwood leaf origin adventitious bud

Abstract

The invention discloses a method for promoting the origin of adventitious buds of cornus florida leaves, and belongs to the technical field of plant tissue culture and propagation. According to the method, adventitious bud culture is carried out on the leaf-derived explants, elements contained in an adopted growth regulator are suitable for metabolism and transportation of carbohydrates in cornus florida, the quality and the propagation coefficient of cornus florida can be improved, and large-scale propagation can be effectively carried out.

Description

TECHNICAL FIELD

[0001] The application relates to a leaf explant adventitious bud promoting method of Cornus florida, and belongs to the technical field of plant tissue culture propagation. BACKGROUND

[0002] Cornus florida is a street tree with beautiful flowers and leaves, and can also be used as a shade tree. Generally, seeds, cuttage and grafting are used for propagation, but the seeds can only be imported and are not easy to purchase, and the price is several thousand yuan per kilogram. The seed propagation offspring is also prone to variation. The survival rate of cuttage can reach more than 80%, but the mother tree is slow-growing, and the branch of the cuttage standard is less. Grafting makes large-scale propagation difficult due to the lack of stock resources. Therefore, tissue culture technology is considered as one of the methods for expanding Cornus florida in addition to conventional propagation, and the leaf is easy to obtain and is the best tissue culture material. SUMMARY

[0003] The application aims to provide a leaf explant adventitious bud promoting method of Cornus florida, which solves the problems of slow propagation speed, small propagation quantity and few effective propagation branches in the prior art by using leaf explant tissue culture technology.

[0004] The technical scheme is as follows: (1) placing the leaf of Cornus florida with the surface upwards on a culture medium to promote callus, and culturing for 4-8 weeks, wherein the culture medium is 1 / 2MS+CPPU 0.1-0.5mg / L+2,4-D 1-10mg / L, and 30g / L of sucrose and 8g / L of agar are additionally added, and the pH value is adjusted to 5.7; (2) culturing the callus into adventitious buds.

[0005] Further, the culture condition of the method is 16 hours of day length, 25±2 DEG C, and 3000 lux of light.

[0006] Further, the step (1) further comprises a sterilization step of the leaf.

[0007] Further, the sterilization step comprises the following steps: soaking in sodium hypochlorite and then washing with sterile water.

[0008] Further, the culture medium used in the step (2) is 1 / 3MS+CPPU 0.5-1mg / L+2,4-D 1-10mg / L, and 30g / L of sucrose and 8g / L of agar are additionally added, and the pH value is adjusted to 5.7.

[0009] Further, the method further comprises the step of collecting the branch of Cornus florida and culturing until the axillary bud grows into a leaf.

[0010] Furthermore, the disinfected branches were placed on a culture medium for axillary bud culture. The culture medium consisted of 1 / 2 MS + CPPU 1~1.5 mg / L + 2,4-D 0.1 mg / L, with 30 g / L sucrose and 8 g / L agar added, and the pH was adjusted to 5.7.

[0011] Further, the disinfection steps are as follows: soak the exogenitals in 75% alcohol for 40 seconds and then discard the alcohol; then soak the exogenitals in 2% sodium hypochlorite for 20 minutes and discard the sodium hypochlorite; and finally rinse three times with sterile water.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The method of this application, through adventitious bud culture of leaf-derived explants, uses growth regulators containing elements suitable for the metabolism and transport of carbohydrates in the body of Cotoneaster multiflora, which can improve the quality and propagation coefficient of Cotoneaster multiflora and can effectively carry out large-scale propagation. Attached Figure Description

[0013] Figure 1 Branches from *Cornus multiflora* (approximately 5 years old, 2 meters tall) were used as explants; Figure 2 The effect of basal culture medium and CPPU concentration; Figure 3 Callus tissue was cultured from the leaves; Figure 4 Callus tissue forms on the leaves; Figure 5 Adventitious buds form on the callus tissue. Detailed Implementation

[0014] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0015] Example 1: Cultivation of axillary buds On a sunny afternoon, branches from a 5-year-old, 2-meter-tall *Cornus multiflora* plant at the base were used as explants. Figure 1 Soak the exogenitals in 75% alcohol for 40 seconds and then discard the alcohol. Soak the exogenitals in 2% sodium hypochlorite for 20 minutes and then discard the alcohol. Rinse three times with sterile water. MS, 1 / 2 MS, N6, and WPM were used as basal media, with sucrose 30 g / L and agar 8 g / L added respectively. The growth regulators chlorpyrifos (CPPU) at concentrations of 0.1–1.5 mg / L (see Table 1) and 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1 mg / L were used. The pH was adjusted to 5.7. Axillary buds were cultured from these explants for 4–8 weeks under the following conditions: 16 hours of daylight, 25 ± 2℃, and 3000 lux light. Contamination occurred during the 4–8 week culture period. After approximately one month, once growth had stabilized, the axillary bud incidence, bud length, and number of leaves were investigated.

[0016] Table 1. Preparation of Basic Culture Media and Growth Regulators Treatment number Basic medium CPPU mg / L 1 MS 0.1 2 0.5 3 1 4 1.5 5 1 / 2MS 0.1 6 0.5 7 1 8 1.5 9 N6 0.1 10 0.5 11 1 12 1.5 13 WPM 0.1 14 0.5 15 1 16 1.5 from Figure 2 As can be seen, the values ​​of treatments 7 and 8 are significantly higher, with the highest axillary bud occurrence rate of 79.3%, bud length of 6.7 cm, and number of leaves of 6.9. The differences are significant using analysis of variance. In particular, the cumulative effect of all indicators in treatment 7 is the best. Therefore, the optimal axillary bud culture medium is determined to be 1 / 2 MS + CPPU 1 mg / L + 2,4-D 0.1 mg / L, with the pH value adjusted to 5.7.

[0017] Example 2: Promoting Healing After the axillary buds sprout leaves, cut off 1-3 leaves, remove the petiole from the leaves, and cut them into 5mm squares to serve as leaf explants. Soak the explants in 2% sodium hypochlorite for 20 minutes and then discard the solution. Rinse three times with sterile water and disinfect. Place the leaf surface upwards in a 20mm deep, 90mm diameter petri dish containing 20ml of water under sterile conditions. Place 6 leaf explants in each petri dish.

[0018] A three-factor, three-level orthogonal experimental design (L9(3)) was used to investigate the formation of callus tissue in the aforementioned leaves. 4 To screen basic culture media and growth regulators, three basic culture media, MS, 1 / 2 MS, and 1 / 3 MS, were used for the experiment. The growth regulators used were CPPU at a concentration of 0.1 mg / L to 1 mg / L, combined with 2,4-D at a concentration of 0.1 to 10 mg / L, with 30 g / L sucrose and 8 g / L agar added to form the basic composition of the culture medium. The pH value was adjusted to 5.7. Detailed information on the culture medium is shown in Table 2.

[0019] Explants were cultured in an artificially lit chamber with 16 hours of daylight, 25±2℃, and 3000 lux for 6–8 weeks. Callus formation on leaves was investigated. The orthogonal experimental design is shown in Table 3.

[0020] Table 2. Levels of influencing factors in orthogonal experimental design (L9 (3)) 4 )) Level Medium A CPPU (mg / L) B 2,4-D (mg / L) C 1 1 / 2MS 0.1 0.1 2 MS 0.5 1 3 1 / 3MS 1 10 Note: 1 / 2MS and 1 / 3MS refer to MS media in which the amounts of KNO3, NH4NO3, and KH2PO4 are reduced to 1 / 2 or 1 / 3. Table 3 Orthogonal Experimental Design Table Experimental combination A B C 1 1 1 1 2 1 2 2 3 1 3 3 4 2 1 2 5 2 2 3 6 2 3 1 7 3 1 3 8 3 2 1 9 3 3 2 Table 4. Analysis of variance for callus tissue Source III type square sum df Mean square F P Medium 6.222 2 3.111 28.000 0.034 CPPU 24.889 2 12.444 112.000 0.009 2,4-D 21.556 2 10.778 97.000 0.010 Error .222 2 0.111 Total 508.000 9 Corrected total 52.889 8 SPSS 24 ANOVA (Table 4) shows that the p-value for the number of callus formations under the influence of culture medium was 0.034, which is less than 0.05, indicating that the culture medium has a significant effect on callus formation. Similarly, the p-values ​​for the number of callus formations under the influence of CPPU and 2,4-D were 0.009 and 0.01, respectively, both less than or equal to 0.01, indicating that hormones CPPU and 2,4-D have a highly significant effect on the number of callus formations. To further screen the optimal hormone concentrations for promoting callus formation, multiple comparisons will be performed on the analysis results.

[0021] Table 5. Multiple comparisons of the effects of culture medium on the number of callus formations. Medium Average number 5% 1% 3 4.5 b B 2 7.7 a A 1 8.9 a A Table 5 shows that the number of callus tissues under the influence of culture medium No. 2 was significantly different from that under the influence of culture medium No. 1 at the 0.05 level but not at the 0.01 level; while the number of callus tissues under the influence of culture medium No. 3 was not significantly different from that under both culture medium No. 1 and No. 2 at the 0.05 and 0.01 levels. Therefore, it can be determined that culture medium No. 1 is the most suitable culture medium for promoting callus formation.

[0022] Table 6. Multiple comparisons of the effect of CPPU on the number of callus formations. CPPU Average number 5% 1% 3 2.4 c C 2 6.1 a A 1 4.7 b B Table 6 shows that the number of callus tissues under CPPU concentration 3 was significantly different from that under concentration 1 at both the 0.05 and 0.01 levels; while the number of callus tissues under concentration 2 was significantly different from that under concentration 3 at both the 0.05 and 0.01 levels. Therefore, it can be determined that CPPU concentration 2 is the optimal concentration for promoting callus growth.

[0023] Table 7. Multiple comparisons of the effect of 2,4-D on the number of callus formations. 2,4-D Average number 5% 1% 1 5 b B 3 8.67 a A 2 7.9 a A Table 7 shows that the number of callus tissues under the influence of concentration 3 of 2,4-D was significantly different from that under concentration 1 at both the 0.05 and 0.01 levels; while the number of callus tissues under the influence of concentration 2 was not significantly different from that under concentration 3 at both the 0.05 and 0.01 levels. Therefore, concentration 3 of 2,4-D can be determined as the optimal concentration for promoting the number of callus tissues formed.

[0024] Based on the above experimental results, the optimal culture medium for promoting callus formation is 1 / 2 MS + CPPU 0.5 mg / L + 2,4-D 10 mg / L, with the addition of sucrose 30 g / L and agar 8 g / L, and the pH adjusted to 5.7.

[0025] Example 3: Promoting Adventitious Buds Through the formation of callus tissue ( Figures 3~5 The formation of adventitious buds is an important pathway for plant formation during tissue culture. The callus tissue formed in Example 2 was cut into 0.5cm × 0.5cm cubes and placed on a culture medium. The same experimental methods as in Tables 2 and 3 were used to screen for the most suitable culture medium for adventitious bud formation. The basic composition of the culture medium was 30g / L sucrose and 8g / L agar. The explants were cultured in an artificially lit room with 16 hours of daylight, 25±2℃, and 3000lux for 6–8 weeks. Bud differentiation was investigated one month after the growth entered a stable phase.

[0026] Table 8. Analysis of variance table for bud differentiation Source III type square sum df Mean square F P Medium 43.556 2 21.778 6.323 0.037 CPPU 300.222 2 150.111 43.581 0.022 2,4-D 226.889 2 113.444 32.935 0.023 Table 8 shows that, according to SPSS 24 variance analysis, the p-value for the number of buds formed under the influence of the culture medium was 0.037, which is less than 0.05, indicating that the culture medium has a significant effect on bud differentiation. Similarly, the p-values ​​for bud differentiation under the influence of CPPU and 2,4-D were 0.022 and 0.023, respectively, both less than 0.05, indicating that the hormones CPPU and 2,4-D have a highly significant effect on bud differentiation.

[0027] Table 9. Multiple comparisons of the effects of culture medium on shoot differentiation Medium Average number 5% 1% 1 5.7 b B 2 3.8 c C 3 9.2 a A Table 9 shows that the bud differentiation (number of buds) under the influence of culture medium No. 3 is significantly superior, while the influence of culture medium No. 2 is minimal.

[0028] Table 10 Multiple comparisons of the effect of CPPU concentration on the number of shoots differentiated. CPPU Average number 5% 1% 1 11.00 bc BC 3 14.67 b B 2 24.67 a A Table 10 shows that the number of shoot differentiations under CPPU concentration 3 was not significantly different from that under concentration 1 at the 0.05 and 0.01 levels; however, the number of shoot differentiations under concentration 2 was significantly different from that under concentrations 1 and 3 at the 0.05 level, but not significantly different at the 0.01 level. Therefore, CPPU concentration 2 can be determined as the optimal concentration for promoting shoot differentiation.

[0029] Table 11 Multiple comparisons of the effects of 2,4-D concentration on the number of explant shoots 2,4-D Average number 5% 1% 1 10.00 b B 3 18.33 ab AB 2 22.00 a A Table 11 shows that the number of bud differentiations under the influence of 2,4-D concentration 3 is significantly different from that under the influence of concentration 1 at the 0.05 level, but not at the 0.01 level; while the number of bud differentiations under the influence of concentration 2 is not significantly different from that under concentration 3 at both the 0.05 and 0.01 levels. Therefore, concentration 2 of 2,4-D can be determined as the optimal concentration for promoting the number of bud differentiations.

[0030] Therefore, 1 / 3 MS was selected as the optimal culture medium. Specifically, 1 / 3 MS + 0.5 mg / L (CPPU) + 1 mg / L (2,4-D) was the best experimental combination for promoting shoot differentiation. Sucrose 30 g / L and agar 8 g / L were also added, and the pH was adjusted to 5.7.

[0031] In summary, the optimal axillary bud culture medium for promoting adventitious buds in *Cornus multiflora* leaves is 1 / 2 MS medium + 1 mg / L CPPU + 0.1 mg / L 2,4-D, with the addition of 30 g / L sucrose and 8 g / L agar, adjusted to pH 5.7, and cultured for 4–8 weeks. The optimal callus formation medium is 1 / 2 MS medium + 0.5 mg / L CPPU + 10 mg / L 2,4-D, with the addition of 30 g / L sucrose and 8 g / L agar, adjusted to pH 5.7, and cultured for 6–8 weeks. The optimal bud differentiation medium is 1 / 3 MS medium + 0.5 mg / L CPPU + 1 mg / L 2,4-D, with the addition of 30 g / L sucrose and 8 g / L agar, adjusted to pH 5.7, and cultured for 6–8 weeks.

Claims

1. A method for promoting the origin of adventitious buds from leaves of *Cornus multiflora*, characterized in that, The method includes the following steps: (1) Place the leaves of Cotoneaster multiflora with the surface facing up on the culture medium to promote callus formation. Culture for 4-8 weeks. The culture medium is 1 / 2 MS + CPPU 0.1-0.5 mg / L + 2,4-D 1-10 mg / L, with 30 g / L sucrose and 8 g / L agar added. The pH value is adjusted to 5.

7. (2) The callus tissue can be cultured as adventitious shoots.

2. The method according to claim 1, characterized in that, The cultivation conditions described are 16 hours of daylight, 25±2℃, and 3000 lux light.

3. The method according to claim 1, characterized in that, Step (1) also includes the step of disinfecting the leaves.

4. The method according to claim 3, characterized in that, The disinfection steps include: soaking in sodium hypochlorite and then rinsing with sterile water.

5. The method according to claim 1, characterized in that, The culture medium used in step (2) is 1 / 3 MS + CPPU 0.5~1 mg / L + 2,4-D 1~10 mg / L, with 30 g / L of sucrose and 8 g / L of agar added. The pH value is adjusted to 5.7, and the culture time is 6~8 weeks.

6. The method according to claim 1, characterized in that, The method also includes the step of collecting branches of *Cornus multiflora* and cultivating them until leaves grow from axillary buds.

7. The method according to claim 6, characterized in that, The sterilized branches were placed on a culture medium for axillary bud culture. The culture medium was 1 / 2 MS + CPPU 1~1.5 mg / L + 2,4-D 0.1 mg / L, with 30 g / L sucrose and 8 g / L agar added. The pH was adjusted to 5.7, and the culture time was 6~8 weeks.

8. The method according to claim 7, characterized in that, The disinfection steps are as follows: soak the exosomes in 75% alcohol for 40 seconds and then discard the alcohol; then soak the exosomes in 2% sodium hypochlorite for 20 minutes and discard the sodium hypochlorite; and finally rinse three times with sterile water.

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