Massive proliferation method for floral gardenia

Abstract

The invention discloses a floral gardenia mass proliferation method which comprises the following steps: (1) collecting and cutting explants, and sterilizing the explants; in the sterilization step, the explants are soaked with alcohol, sodium hypochlorite and a mixed pesticide solution in sequence; the mixed pesticide solution is a mixed solution of zinc thiazole, lentinan and zhongshengmycin; the soaking time of the mixed pesticide solution is 10-15 minutes; and (2) carrying out primary culture, subculture and rooting culture on the middle part of the branch of the sterilized explant by using a culture medium. By optimizing the explant sterilization method of the floral gardenia and screening the culture media for the primary culture, the subculture and the rooting culture, the invention customizes a cultivation method which can quickly and massively cultivate the floral gardenia and is excellent in various aspects such as the number of starting roots, the length of each root, the rooting rate and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture and relates to a method for mass proliferation of variegated gardenias. Background Art

[0002] Variegated Gardenia (Gardenia jasminoides 'Variegata') is a colorful plant in the Rubiaceae family. Its large, vibrant leaves are fresh and bright, and it is cold- and heat-tolerant, making it suitable for gardening or potted plants. Cuttings are a common propagation technique, but these are scarce and cannot be supplied in large quantities. Therefore, tissue culture has become the fastest and most effective method for propagating variegated gardenia. Summary of the Invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a method for cultivating gardenia jasminoides that can be extensively propagated.

[0004] Technical solution: A method for mass proliferation of variegated gardenias of the present invention comprises the following steps:

[0005] (1) collecting and cutting the explants, and sterilizing the explants; the sterilization step comprises sequentially soaking the explants with alcohol, sodium hypochlorite, and a mixed pesticide solution; the mixed pesticide solution is a mixed solution of zinc thiazole, lentinan, and zhongshengmycin; and the soaking time of the mixed pesticide solution is 10 to 15 minutes.

[0006] (2) Use culture medium to perform primary culture, subculture and rooting culture on the middle part of the branches of the sterilized explants.

[0007] Furthermore, the amount of zinc thiazole in step (1) is 0.12 g / 400 mL, the amount of lentinan is 1.1 g / 400 mL, and the amount of zhongshengmycin is 1.1 g / 400 mL.

[0008] Furthermore, the alcohol soaking time in step (1) is 40 seconds, and the sodium hypochlorite soaking time is 10 minutes.

[0009] Furthermore, the culture medium used for the primary culture in step (2) is B5+0.5 mg / L 6-BA+0.2 mg / L NAA+6.5 g / L agar+35 g / L sucrose, with a pH of 5.6.

[0010] Furthermore, the culture medium used for the subculture in step (2) is B5+1-2 mg / L 6-BA+0.1-1 mg / L NAA+30 g / L sucrose+6.5 g / L agar, with a pH of 5.8.

[0011] Furthermore, the culture medium used for the rooting culture in step (2) is B5+1-2 mg / L 6-BA+0.1-1 mg / L 1 BA+30 g / L sucrose+6.5 g / L agar, with a pH of 5.8.

[0012] Furthermore, the step of collecting and cutting the explants in step (1) is as follows: on the afternoons of four consecutive sunny days, collect strong branches free of pests and diseases as explants; remove leaves from the stems, leaving 1 to 1.5 cm above the buds and 1 to 2 cm below the buds; and wash away dust and dirt from the stems with laundry detergent.

[0013] Furthermore, the strong branches have a stem diameter of 0.2 to 0.5 cm.

[0014] Furthermore, the method also includes the step of fertilizing the mother plants.

[0015] Furthermore, the mother plant fertilization step is: strengthening fertilizer and water management for the plants from which explants are to be collected in the autumn and winter of the previous year.

[0016] Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: by optimizing the explant sterilization method of variegated gardenia and screening the culture medium used for primary culture, subculture and rooting culture, the present invention customizes a cultivation method for variegated gardenia that can quickly and massively cultivate roots, with excellent root number, uniform root length, rooting rate and other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Callus incidence and bud germination in subculture;

[0018] Figure 2 The elongation and proliferation rate of buds in the callus-generating area during subculture;

[0019] Figure 3 The effect of culture medium on root number and average root length during rooting culture;

[0020] Figure 4 Effect of rooting medium on rooting rate. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0022] Example 1

[0023] (1) Fertilization of mother plants: During the autumn and winter of the previous year (late October to November), strengthen fertilizer and water management for the plants from which explants are to be collected (the mother plants are ground-planted seedlings or potted seedlings). This will increase the number of strong branches and improve the branches' resistance to disease and insects. Specifically, apply 50g of sheep manure plus 5g of compound fertilizer (N:P:K ratio of 20:20:20) to each seedling, and apply along the periphery of the plant or along the edge of the pot. Water the plants thoroughly when they are dry.

[0024] (2) Collection of exosomes: In the afternoons (13:00-15:00) of four consecutive sunny days, strong branches (stem diameter of 0.2-0.5 cm) without pests and diseases were collected as exosomes.

[0025] (3) Excision of the explants: Remove the leaves from the stem, leaving 1-1.5 cm above the bud and 1-2 cm below the bud. Do not leave the petiole. Wash the stem clean of dust and dirt with laundry detergent.

[0026] (4) Sterilization: Soak in 75% alcohol for 40 seconds, then soak in 2% sodium hypochlorite for 10 minutes, and then soak in a mixed pesticide solution for 10 to 25 minutes (this is mainly for sterilization testing, and the culture medium is used to culture the contaminated strains after sterilization).

[0027] Table 1 Effect of different treatment times of culture medium sterilization on preservation rate

[0028]

[0029] In Table 1, a combination of commonly used pesticides is used for exosome sterilization, supplemented by alcohol and sodium hypochlorite soaking, to solve the problem of incomplete sterilization using only alcohol and sodium hypochlorite. The use of a combination of pesticides for sterilization solves the problem of using mercuric chloride (a highly toxic agent that has been banned from the market) in the past.

[0030] Zinc thiazole is a drug for preventing and treating bacteria, Lentinan can resist viruses, and Sinobiotics is a broad-spectrum antibiotic. According to the instructions, use the corresponding concentration for soaking for 10 to 25 minutes.

[0031] The results showed that soaking for 15 minutes had the best effect, with the explant preservation rate (calculated as (number of tests - number of losses)%) reaching 80%. After soaking for 20 to 25 minutes, the number of explants killed (the killing number is the direct damage to the explants by the agent, which is manifested as the explants having no mycelium, buds being rigid and not germinating) increased significantly, and the preserved explants not germinating would cause the subculture to be suppressed.

[0032] (5) Primary culture: The culture medium and conditions for primary culture were optimized using the orthogonal test (L9(34)). Three culture media were used: MS, B5, and WPM; hormones used were 6-BA (0.2 mg / L, 0.5 mg / L, 0.8 mg / L) and NAA (0.4 mg / L, 0.2 mg / L, 0.1 mg / L). At the same time, a plant was divided into different parts (upper part, middle part, and base).

[0033] 6.5g / L agar, 35g / L sucrose, and pH 5.6 were also added to the culture medium, which was then packaged and sterilized under high pressure. Under sterile conditions, the sterile seedlings were divided into independent plants, each divided into three parts: the upper section, the middle section, and the base (each branch can be divided into three parts: the upper section, the middle section, and the base, and the sections are generally divided equally), and then inoculated into the subculture medium. After inoculation, they were placed in a culture room with a temperature of 25°C, a light intensity of 3000Lx, and a light length of 12h / d. Every 7 days (record once every 7 days, record 5 times for about 1 month), observe and record the number of new buds and leaves.

[0034] Table 2 Orthogonal test factors and levels

[0035]

[0036]

[0037] According to the requirements of the three-factor three-level orthogonal experiment, the culture medium and stem segment screening combinations are shown in Table 3:

[0038] Table 3 Three-factor three-level combination table

[0039]

[0040] Range Analysis of the Effect of Culture Medium on the Number of New Shoots: The range (R) of the three factors clearly indicates the degree of influence of culture medium, hormone concentration, and stem segment on the number of new buds (Table 4). Table 4 shows that the order of influence of the three factors on the number of new buds is culture medium > stem segment > 6-BA:NAA, indicating that the type of culture medium has the greatest impact on the number of buds, followed by the location of the sterile seedlings, and finally the growth regulator. The mean values ​​(Ki) of each factor indicate that for culture medium type: A1 > A2 > A3; for hormone concentration: B2 > B3 > B1; and for stem segment location: C2 > C3 > C1.

[0041] Table 4 Range analysis of the number of new buds

[0042]

[0043] Variance analysis of the effect of culture medium on leaf number: Compared with the simple and intuitive range analysis that can see the optimal combination, SPSS variance analysis can more accurately determine whether there is a difference between the three factors and the number of leaf growth. As shown in Table 5, the F of culture medium type is 32.987, P = 0.000 < 0.01, indicating that the culture medium type has the most significant effect on leaf growth; the F of hormone concentration is 3.263, 0.01 < P = 0.043 < 0.05, indicating that the hormone concentration has a significant effect on leaf growth; and the F of different parts of the stem segment is 2.130, P = 0.123 > 0.05, so the stem segment position has no significant effect on leaf growth.

[0044] Table 5 Analysis of variance of leaf number results

[0045] source Type III Sum of Squares degrees of freedom mean square F Significance Revised model 1650.800a 6 275.133 12.793 .000 intercept 1159.211 1 1159.211 53.902 .000 A 1418.822 2 709.411 32.987 .000 B 140.356 2 70.178 3.263 .043 C 91.622 2 45.811 2.130 .125 error 1784.989 83 21.506 total 4595.000 90 Revised total 3435.789 89

[0046] aR² = .480 (adjusted R² = .443)

[0047] Based on the above results, the optimal combination is: A1B2C2, that is, B5+0.5mg / L 6-BA+0.2mg / LNAA+middle part of the branch.

[0048] (6) Subculture: Use B5 as the basic culture medium for subculture, with a growth regulator concentration of 0.25-2 mg / L 6-BA and 0-1 mg / L NAA (see Table 6). Add 30 g / L sucrose and 6.5 g / L agar to the culture medium, and adjust the pH to 5.8. Screen for the most suitable culture medium and growth regulator ratio. After one month, investigate and analyze the experimental data.

[0049] Table 6 Ratio of subculture medium and growth regulator

[0050]

[0051]

[0052] By statistical analysis of the test results, see Figure 1 The results showed that the highest callus incidence rate was 93.1% in treatment No. 11 and the lowest was 3.5% in treatment No. 1. The highest bud germination rate was 69.3% in treatment No. 11 and the lowest was 47.7% in treatment No. 4.

[0053] from Figure 2 It can be seen that the highest callus area in the subculture is treatment 11, which is 5.2 mm 2 , the lowest is treatment No. 1 which is 0mm 2 The highest bud elongation was in treatment 11, and the lowest was 1.3 cm in treatment 1. The variance analysis showed that there was no significant difference in the multiplication rate.

[0054] From the above, it can be concluded that treatment 11 is the most suitable culture medium for subculture, which is B5+6-BA 1mg / L+NAA0.1 mg / L+sucrose 30g / L+agar 6.5g / L, with a pH of 5.8.

[0055] (7) Rooting Culture: Based on primary and secondary culture, Gardenia jasminoides is a plant that readily forms callus and buds. Therefore, the basic medium for rooting culture was B5, with growth regulators at concentrations of 0.25-2 mg / L 6-BA and 0.001-1 mg / L IBA (see Table 7). Sucrose (30 g / L) and agar (6.5 g / L) were added to the culture medium, and the pH was adjusted to 5.8. One month later, the experimental data were collected and analyzed.

[0056] Table 7 Ratio of rooting medium and growth regulator

[0057]

[0058] from Figure 3 It can be seen that the indexes of the two curves of root number and mean root length are higher between treatment 11 and treatment 14, and through variance analysis, the type of culture medium has a significant effect on the increase of root number and mean root length.

[0059] from Figure 4 As can be seen from the data, the rooting rate was highest in the 1mg / L 6-BA concentration range, 18.13% higher than the 0.25mg / L 6-BA concentration range. Furthermore, analysis of variance showed that the type of culture medium had a significant effect on the rooting rate.

[0060] The rooting medium consists of B5, 1 mg / L 6-BA, 0.1-1 mg / L IBA, 30 g / L sucrose, 6.5 g / L agar, and a pH of 5.8. (In this medium, IBA is a range, a result of comprehensively considering the effects of 6-BA and IBA, excluding the section with the lowest 6-BA effect.)

[0061] Through the above experiments, the tissue culture of Gardenia jasminoides was carried out under the best culture conditions from sterilization before primary culture to rooting culture. The sterilization effect was soaked in 75% alcohol for 40 seconds, then soaked in 2% sodium hypochlorite for 10 minutes, and then soaked in a mixture of 0.12g / 400mL of zinc thiazole + 1.1g / 400mL of lentinan + 1.1g / 400mL of zhongshengmycin for 15 minutes; the primary culture medium was B5+0.5mg / L 6-BA+0.2mg / LNAA+the middle part of the branch, added with 6.5g / L agar, 35g / L sucrose, pH 5.6, the temperature of the culture room was 25℃, the light intensity was 3000Lx, and the light length was 12h / d.

[0062] The subculture medium was B5 + 1 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 6.5 g / L agar, with a pH of 5.8. The rooting medium was B5 + 1 mg / L 6-BA + 0.1-1 mg / L 1 BA + 30 g / L sucrose, 6.5 g / L agar, with a pH of 5.8.

Claims

1. A method for mass proliferation of variegated gardenia, characterized in that: The following steps are involved: (1) collecting and cutting the explants, and sterilizing the explants; the sterilization step comprises sequentially soaking the explants in alcohol, sodium hypochlorite, and a mixed pesticide solution; the mixed pesticide solution is a mixed solution of zinc thiazole, lentinan, and zhongshengmycin; and the soaking time of the mixed pesticide solution is 10 to 15 minutes; (2) Use culture medium to perform primary culture, subculture and rooting culture on the middle part of the branches of the sterilized explants.

2. The method for mass multiplication of variegated gardenia according to claim 1, wherein: The amount of zinc thiazole described in step (1) is 0.12 g / 400 mL, the amount of lentinan is 1.1 g / 400 mL, and the amount of zhongshengmycin is 1.1 g / 400 mL.

3. The method for mass proliferation of variegated gardenia according to claim 1, characterized in that: The alcohol soaking time described in step (1) is 40s, and the sodium hypochlorite soaking time is 10min.

4. The method for mass proliferation of variegated gardenia according to claim 1, characterized in that: The culture medium used for the primary culture described in step (2) is B5+0.5mg / L 6-BA+0.2mg / L NAA+6.5g / L agar+35g / L sucrose, with a pH of 5.6; the culture temperature is 25°C, the light intensity is 3000Lx, and the light length is 12h / d.

5. The method for mass proliferation of variegated gardenia according to claim 1, characterized in that: The culture medium used for the subculture described in step (2) is B5+1-2 mg / L 6-BA+0.1-1 mg / L NAA+30 g / L sucrose+6.5 g / L agar, with a pH of 5.8; the culture temperature is 25°C, the light intensity is 3000 Lx, and the light length is 12 h / d.

6. The method for mass multiplication of variegated gardenia according to claim 1, characterized in that: The culture medium used for the rooting culture in step (2) is B5+1-2 mg / L 6-BA+0.1-1 mg / L IBA+30 g / L sucrose+6.5 g / L agar, with a pH of 5.8; the culture temperature is 25° C., the light intensity is 3000 Lx, and the light length is 12 h / d.

7. The method for mass multiplication of variegated gardenia according to claim 1, characterized in that: The steps for collecting and cutting explants in step (1) are as follows: on the afternoons of four consecutive sunny days, collect strong branches free of pests and diseases as explants; remove leaves from the stems, leaving 1-1.5 cm above the buds and 1-2 cm below the buds; and clean the stems from dust and dirt with laundry detergent.

8. The method for mass multiplication of variegated gardenia according to claim 7, characterized in that: The strong branches have a stem diameter of 0.2 to 0.5 cm.

9. The method for mass multiplication of variegated gardenia according to claim 1, characterized in that: The method further comprises the step of fertilizing the mother plants.

10. The method for mass multiplication of variegated gardenia according to claim 9, characterized in that: The mother plant fertilization and cultivation steps are: strengthening fertilizer and water management on the plants from which explants are to be collected in the autumn and winter of the previous year.

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