Method for massively propagating lateral buds of delphinium ajacis
Through the tissue culture method of delphinium lateral buds, using specific culture medium and conditions for primary, secondary and rooting culture, the problem of genetic instability in delphinium cultivation has been solved, and rapid and stable seedling propagation has been achieved to meet market demand.
Patent Information
- Application Number
- CN202511031057.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-25
- Publication Date
- 2025-09-23
AI Technical Summary
There are problems in the cultivation of delphinium, such as genetic instability, unstable flowering period and unfavorable overwintering seedlings, which lead to low production yield. Existing tissue culture proliferation methods are difficult to achieve large-scale proliferation.
Delphinium lateral buds were used as explants, and primary culture, subculture and rooting culture were carried out by tissue culture. Specific culture media and conditions were used for rapid propagation, including 1/3MS+6-BA 1.5 mg/L+NAA 0.01 mg/L, 1/2MS+6-BA 2 mg/L with added fructose, and 1/2MS+IBA 0.1 mg/L culture media. The pH value was controlled at 5.6, the light exposure was 16 hours per day, and the operation was performed after sterilization.
The rapid propagation of delphinium seedlings with stable traits was achieved to meet market demand, avoid the problem of unstable traits, and improve production efficiency.
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Figure CN120678021A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for mass propagation of delphinium lateral buds, and belongs to the technical field of agricultural plant tissue culture propagation. Background Art
[0002] Delphinium (Consolida ajacis) has seen increasing demand as a cut flower in recent years. Its wide variety of flower colors and shapes, combined with its rich, vibrant colors, has made it highly popular. Many of the varieties currently on the market are derived from variants grown in production gardens. Vegetative propagation, particularly tissue culture, is an effective method for achieving large-scale proliferation of these variants.
[0003] Delphiniums are a member of the Ranunculaceae family, native to dry, cold regions. Approximately 200 species have been identified in the Northern Hemisphere. Currently, most cultivated varieties are cultivated in Europe and the United States, resulting in high production and commercial value. Delphiniums, with their predominantly blue flowers and cut flowers reaching lengths exceeding two meters, have become an essential decorative element for conferences and events in recent years.
[0004] However, several challenges in the cultivation of delphiniums hinder its widespread adoption. The most significant issue is that all varieties cultivated domestically are genetically unstable, a major factor contributing to low production yields. This variable flowering period results in significant production variations, with some varieties taking up to three months to bloom, hindering planned production.
[0005] Furthermore, current greenhouse-grown varieties that flower from March to May require overwintering, which is detrimental to delphiniums, which naturally prefer cooler climates. To address these issues, effective tissue culture propagation of high-quality flowering individuals is essential. Because field-grown plants can only multiply about 20-fold annually, cultivating practical new varieties is difficult.
[0006] Therefore, it is feasible to use biological methods to study the proliferation of delphiniums through tissue culture. To date, there have been attempts to culture delphiniums, but due to problems such as the culture site, culture conditions, and culture medium composition, there have been no successful examples of large-scale proliferation of flowering plants. Summary of the Invention
[0007] Purpose of the invention: The purpose of the present invention is to provide a method for mass propagation of delphinium using lateral buds as explants in tissue culture.
[0008] Technical solution: A method for mass propagation of delphinium lateral buds of the present invention comprises the following steps:
[0009] (1) Select delphiniums that have completed their first flowering, collect the lateral buds at their base as explants, remove excess leaves, clean and sterilize, and remove the tissues containing the growth point and several leaf primordia;
[0010] (2) The treated explants are subjected to primary culture, subculture, rooting culture and seedling hardening;
[0011] The primary culture medium is 1 / 3MS+6-BA 1.5 mg / L+NAA 0.01 mg / L, with 3% sucrose and 3% agar added, and a pH of 5.6.
[0012] Furthermore, the culture medium for the subculture is 1 / 2MS+6-BA2-4 mg / L or B5+6-BA2 mg / L.
[0013] Furthermore, 3% fructose or maltose, 3% agar, and pH 5.6 are added to the subculture.
[0014] Furthermore, the rooting culture medium is 1 / 2MS+IBA0.1-1 mg / L, with 3% sucrose and 3% agar added, and pH 5.6.
[0015] Furthermore, the number of subcultures is 1 to 3 times.
[0016] Furthermore, the culture conditions for the primary culture, subculture and rooting culture are 25° C. and 16 hours of lighting per day.
[0017] Furthermore, the sterilization step is to soak the explant in 70% ethanol and sodium hypochlorite once, and then wash it with sterile water until the chlorine odor disappears.
[0018] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: It utilizes lateral buds of delphinium as explants to produce stable delphinium seedlings through tissue culture, rapidly expanding the breeding base of delphinium. This prevents the phenomenon of unstable traits that occurs during sexual propagation, allowing the breeding speed of delphinium to meet market demand. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Effects of growth regulator ratio of 1 / 3MS culture medium on bud and callus formation. DETAILED DESCRIPTION
[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings.
[0021] Example 1 Investigation of primary culture medium
[0022] Step 1 Test materials: Delphiniums that have completed their first flowering are selected from the greenhouse, and the small lateral buds growing from their bases are used as explant materials, including the underground parts below 1 cm. A maximum of 14 lateral buds can be collected from one plant.
[0023] Step 2 Material preparation: Wash with tap water and remove excess leaves.
[0024] Step 3: Sterilization: Disinfect the exosome with 70% ethanol for 10 seconds, soak and stir in sodium hypochlorite with 0.5% available chlorine for 20 minutes, then change the sterilized water three times and rinse until the chlorine odor disappears. It is ready for aseptic operation.
[0025] Step 4: From the sterilized sample, tissue containing the growth point and several leaf primordia is removed under a stereomicroscope (removed with 1 to 2 leaf primordia attached to the top of the stem (0.3 to 0.5 mm)), and placed on the culture medium at a density of 1 stem top per container (20 mL of culture medium) for primary culture medium screening.
[0026] The basal culture medium of the primary culture medium is MS, 1 / 2MS, 1 / 3MS, and WPM, to which 3% sucrose and 3% Gelrite (a plant gel) are added respectively, and the pH is adjusted to pH 5.6.
[0027] The light intensity was 2200 Lx for 16 hours per day at 25°C. After 4 weeks, the survival rate, bud germination rate, and callus formation ability of the regenerated plants were investigated.
[0028] Table 1 Exozoite status in various culture media during primary culture
[0029]
[0030] As shown in Table 1, among the four primary basic culture media, the survival rate of 1 / 3MS medium was the highest at 95%, and WPM was the lowest at 58%. The bud germination rate of 1 / 3MS was the highest at 91%, and the ability to form callus tissue was also the strongest.
[0031] Furthermore, the contents of 6-BA and NAA in the 1 / 3MS primary culture medium were investigated, using four concentration gradients: 6-BA concentrations of 0, 0.5, 1.5, and 2 mg / L, and NAA concentrations of 0, 0.01, 0.1, and 1 mg / L (see Table 2). There were a total of 16 treatments, and 3% sucrose and 3% agar were added to each treatment, and the pH was adjusted to 5.6. After 30 days, the elongation of the buds, the germination rate of the buds, the incidence rate of callus, and the area of callus occurrence were investigated.
[0032] Table 2 Combination ratios of 6-BA and NAA in 1 / 3MS primary culture medium
[0033]
[0034]
[0035] from Figure 1 It can be seen that the optimal ratio of 6-BA and NAA in 1 / 3MS primary culture medium is treatment 10 1 / 3MS + 6-BA 1.5 mg / L + NAA 0.01 mg / L. From the variance analysis results of bud elongation, bud germination rate, callus incidence rate, callus area, etc., there are significant differences among the treatments and among the indicators.
[0036] Example 2 Investigation of Subculture Medium
[0037] For subculture, plants obtained from primary culture were excised at a height of approximately 5 mm from the base, arranged to have one bud per node, and plated at a density of five plants per container (50 mL of culture medium). The basal medium consisted of 1 / 2 MS, 1 / 3 MS, and B5, with 1-4 mg / L 6-BA, 0 mg / L NAA, and 3% agar, with a pH of 5.6. The culture temperature was 25°C with a light intensity of 2200 Lx for 16 hours per day. The plants were inspected and counted every four weeks, and subculture continued.
[0038] Table 3 6-BA in subculture medium
[0039]
[0040] Each cutting is done when the plant from the previous generation reaches the top of the bottle. Subculture can continue for many generations (i.e., multiple cuttings). When cutting, remove the tissue culture bottle filled with plants from the culture rack. In a sterile inoculation room, remove the plant from the bottle, remove the leaves, and leave 1 cm above each node and 1.5 cm below each node. Inoculate the new bottle.
[0041] As can be seen from Table 3, the concentration of 6-BA added to the secondary culture medium had significant differences in proliferation rate, with the highest average proliferation rate of 3.4 when the concentration was 2 mg / L in the basic culture medium 1 / 2MS.
[0042] The effects of different sugars on subculture were further investigated in a medium containing 1 / 2 MS and 2 mg / L 6-BA. 3% sucrose, maltose, glucose, fructose, or mannitol were added. Statistics were collected after four weeks of culture.
[0043] Table 4 Effects of adding different sugars in subculture
[0044]
[0045]
[0046] As shown in Table 4, different sugar substances have different effects on the proliferation rate and branch length during subculture. The proliferation rate of fructose is the highest, reaching 3.6 times, and the average branch length is 9.6 cm. The proliferation rate of mannitol is the lowest, at 1.7 times, and the average branch length of sucrose is 3.7 cm.
[0047] In summary, the best combination of culture medium for several generations was 1 / 2MS+6-BA2 mg / L with 3% fructose and 3% agar added, and the pH was adjusted to pH 5.6.
[0048] Example 3 Investigation of rooting culture medium
[0049] For rooting, all subcultured plants were removed and plated at a density of five plants per container (50 mL of medium) on a rooting medium. The basal medium consisted of 1 / 2 MS, 1 / 3 MS, and B5, with 0-1 mg / L IBA, 3% sucrose, and 3% agar, and the pH was adjusted to 5.6.
[0050] Table 5 Effect of IBA concentration in rooting medium on rooting
[0051]
[0052] After 6 weeks of cultivation at 25°C with 2200 Lx of light for 16 hours per day, the rooting rate and average root number were assessed. As shown in Table 5, the rooting rate was highest at all concentrations in 1 / 2 MS minimal medium, with the highest at 85.4% at 0.1 mg / L IBA.
[0053] Results: The optimal culture conditions determined from this experiment were that the primary culture medium consisted of 1 / 3 MS with 1.5 mg / L 6-BA and 0.01 mg / L NAA, supplemented with 3% sucrose and 3% agar, with a pH of 5.6, achieving the best culture efficiency. The secondary culture medium consisted of 1 / 2 MS with 2 mg / L 6-BA, supplemented with 3% fructose and 3% agar, with a pH of 5.6. The rooting medium consisted of 1 / 2 MS with 0.1 mg / L IBA, supplemented with 3% sucrose and 3% agar, with a pH of 5.6.
[0054] Hardening of seedlings: Remove the culture medium from the roots of the rooted seedlings, wash them, be careful not to break the stems during the operation, plant them in a peat + perlite matrix, cover with a shade net, spray frequently in the early stage, and manage them normally after one month.
Claims
1. A method for mass propagation of delphinium lateral buds, characterized in that: The following steps are involved: (1) Select delphiniums that have completed their first flowering, collect the lateral buds at their base as explants, remove excess leaves, clean and sterilize, and remove the tissues containing the growth point and several leaf primordia; (2) The treated explants are subjected to primary culture, subculture, rooting culture and seedling hardening; The primary culture medium is 1 / 3MS+6-BA 1.5 mg / L+NAA 0.01 mg / L, with 3% sucrose and 3% agar added, and a pH of 5.
6.
2. The method for mass propagation of delphinium lateral buds according to claim 1, characterized in that: The culture medium for the subculture is 1 / 2MS+6-BA 2-4 mg / L or B5+6-BA 2 mg / L.
3. The method for mass propagation of delphinium lateral buds according to claim 2, characterized in that: 3% fructose or maltose and 3% agar are further added to the subculture, and the pH value is 5.
6.
4. The method for mass propagation of delphinium lateral buds according to claim 1, characterized in that: The rooting culture medium is 1 / 2MS+IBA 0.1-1 mg / L, added with 3% sucrose and 3% agar, and has a pH of 5.
6.
5. The method for mass propagation of delphinium lateral buds according to claim 1, characterized in that: The number of subcultures is 1 to 3 times.
6. The method for mass propagation of delphinium lateral buds according to claim 1, characterized in that: The culture conditions for the primary culture, subculture and rooting culture are 20° C. and 16 hours of lighting per day.
7. The method for mass propagation of delphinium lateral buds according to claim 1, characterized in that: The sterilization step comprises soaking the explants in 70% ethanol and sodium hypochlorite once, and then washing with sterile water until the chlorine odor disappears.