Rapid propagation method of ixora chinensis
By using specific culture medium formulations and treatment methods, the problem of low propagation efficiency of Ixora has been solved, enabling rapid propagation and new variety development of Ixora, and providing efficient techniques for inducing clustered buds and rooting.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- MINNAN NORMAL UNIV
- Filing Date
- 2024-08-30
- Publication Date
- 2026-07-21
AI Technical Summary
In existing technologies, the propagation efficiency of Ixora is low, making it difficult to achieve rapid propagation and the breeding of new varieties.
Using specific culture medium formulations and treatment methods, including shoot induction medium (MS + 4 mg/L 6-BA + 1 mg/L NAA + 1 mg/L TDZ) and cluster shoot induction medium (MS + 0.5 mg/L 6-BA + 0.5 mg/L NAA), combined with suitable light and temperature conditions, Ixora branches were treated and cultured to induce cluster shoots and rooting.
It sprouts within 15 days, and the rooting rate reaches 85% within 30 days. The average number of buds in a cluster reaches 20, and the plant is in good condition, providing a theoretical basis for the rapid propagation and breeding of Ixora.
Smart Images
Figure CN118947543B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of Ixora breeding, specifically to a method for rapid propagation of Ixora. Background Technology
[0002] Ixora chinensis Lam, also known as White Sunflower, Fairy Flower, Mountain Lily, etc., is a plant of the Rubiaceae family and the Ixora genus.
[0003] Ixora, with its vibrant colors and blooming period from March to December, is a relatively short plant with high ornamental value, widely used in landscaping and the cut flower market. The induction of clumping buds is an important step in plant tissue culture and is of great significance for the propagation of Ixora and the development of new varieties. Summary of the Invention
[0004] The purpose of this invention is to provide a rapid propagation method for Ixora chinensis and to establish an efficient Ixora chinensis bud induction system, providing a reference for the rapid propagation and breeding of Ixora chinensis.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] A method for rapid propagation of Ixora includes the following steps:
[0007] 1) Select healthy, semi-lignified Ixora branches with terminal buds and mature leaves from the current year as explants. Cut off the entire branch with pruning shears (explants are generally collected on a sunny morning between 9 and 11 am).
[0008] 2) After pruning the leaves of the Ixora chinensis branches, leave 3-4 cm below the terminal bud and place them in a 500 mL culture bottle with 4-5 branches per bottle. On a clean bench sterilized with ultraviolet light, first soak the explants in 75% ethanol for 30±2 seconds, then rinse them once with sterile water. Add 2% sodium hypochlorite solution to cover the explants, then add 1‰-2‰ Tween 80 and sonicate at 500W in the dark for 8-10 minutes to ensure thorough sterilization. Finally, rinse them 2-3 times with sterile water.
[0009] 3) Transfer the culture flask to a sterile laminar flow hood, fill it with 2% sodium hypochlorite solution, and soak it in the dark for 5-8 minutes. Gently shake the Erlenmeyer flask during the soaking process to ensure thorough sterilization. Finally, rinse it 2-3 times with sterile water.
[0010] 4) Place the branches on sterile filter paper and let them dry completely. Then, use sterile forceps and a scalpel to completely remove the oxidized and blackened parts of the branches. Make a slanted cut at the lower morphological end of the branches. During the inoculation process, inoculate the branches at a 45° angle into the bud induction medium.
[0011] The method for preparing the budding induction medium is as follows: 30 g / L sucrose and 6.5 g / L agar are added to MS medium to obtain the basic medium, and then 4 mg / L 6-benzylaminopurine (6-BA), 1 mg / L naphthaleneacetic acid (NAA), and 1 mg / L thiabendazole (TDZ) are added. Finally, the pH of the medium is adjusted to 5.8.
[0012] 5) After sealing the inoculated culture bottles, place them in an environment with a temperature of 27℃±1℃, a light intensity of 1000lx-1500lx, and a light duration of 16 hours / day for 15 days.
[0013] 6) After sterilizing the culture bottle with 75% alcohol, place it on a sterile operating table. Use sterile forceps to pick up the branch and place it on sterile filter paper. Use a sterile scalpel to cut off the tender new terminal bud of the branch and make an oblique cut at its morphological lower end. Inoculate the branch at a 45° angle into the cluster bud induction medium.
[0014] The method for preparing the bud induction medium is as follows: 30 g / L sucrose and 6.5 g / L agar are added to MS medium to obtain the basal medium, then 0.5 mg / L 6-BA and 0.5 mg / L NAA are added, and finally the pH of the medium is adjusted to 5.8.
[0015] 7) After sealing the inoculated culture flasks, place them in an environment with a temperature of 27℃±1℃, a light intensity of 1000lx-1500lx, and a light duration of 16 hours / day for 30 days.
[0016] 8) Take the clustered buds and divide them into individual buds. Transfer them into rooting medium and culture them under the conditions of temperature 27℃±1℃, light intensity 1000lx-1500lx, and light time 16 hours / day for 30 days. Calculate the rooting rate.
[0017] The rooting induction medium is prepared by adding 30 g / L sucrose and 6.5 g / L agar to 1 / 2 MS medium to obtain the basal medium, then adding 3 mg / L NAA, and finally adjusting the pH of the medium to 5.8.
[0018] This invention studies the culture conditions for inducing shoot clusters in Ixora chinensis, including the selection of culture medium, the concentration of plant growth regulators, the type of explant and treatment methods, etc., and provides a feasible culture system for inducing shoot clusters in Ixora chinensis. It provides a theoretical basis and technical support for the propagation and breeding of new varieties of Ixora chinensis, and at the same time, it also provides a reference for the study of shoot cluster induction in other plants.
[0019] The present invention adopts the above technical solution, and its beneficial effects are as follows:
[0020] 1. Ixora branches can sprout in about 15 days when cultured in bud-forming medium (MS + 4 mg / L 6BA + 1 mg / L NAA + 1 mg / L TDZ);
[0021] 2. The number of shoots generated when the shoots were transferred to the shoot induction medium (MS + 0.5 mg / L 6BA + 0.5 mg / L NAA) was on average 20, and the plants were robust and green.
[0022] 3. The clustered shoots were transferred to rooting induction medium (1 / 2 MS + 3 mg / L NAA) and cultured for 30 days. The rooting rate reached 85% and the root system was well-developed and robust.
[0023] 4. This invention is beneficial to improving the induction rate and rooting rate of Ixora chinensis bud clusters, increasing the number of individual plants, and providing a reference for the rapid propagation and breeding of Ixora chinensis. Attached Figure Description
[0024] Figure 1 The effect of different TDZ concentrations in culture medium on the induction of shoot clusters in Ixora chinensis.
[0025] Figure 2 The effect of different 6-BA concentrations in culture medium on the induction of shoot clusters in Ixora chinensis.
[0026] Figure 3 Inducing rooting in Ixora. Detailed Implementation
[0027] Example 1
[0028] Screening of bud induction culture medium
[0029] 1) On a clear morning between 9 and 11 a.m., select a healthy, semi-lignified Ixora branch with terminal buds and mature leaves, and cut the entire branch off with pruning shears.
[0030] 2) After pruning the leaves of the Ixora branches, leave 3-4 cm below the terminal bud and place them in 500mL Erlenmeyer flasks, with 4-5 branches in each flask. On a clean bench sterilized with ultraviolet light, first soak the explants in 75% ethanol for 30 seconds, then rinse them once with sterile water. Add 2% sodium hypochlorite solution to cover the explants, then add 1‰-2‰ Tween 80. Transfer the explants to a laboratory ultrasonic cleaner and sonicate at 500W in the dark for 10 minutes to ensure thorough sterilization. Finally, rinse them three times with sterile water.
[0031] 3) Transfer the Erlenmeyer flask to a sterile laminar flow hood, fill it with 2% sodium hypochlorite solution, and soak it in the dark for 8 minutes. Gently shake the Erlenmeyer flask during the soaking process to ensure thorough disinfection. Finally, rinse it 3 times with sterile water.
[0032] 4) Place the branches on sterile filter paper and let them dry completely. Then, use sterilized tweezers and a scalpel to completely remove the oxidized and blackened parts of the branches. Make a slanted cut at the lower morphological end of the branches. During the inoculation process, inoculate the branches at a 45° angle into the bud induction medium.
[0033] The method for preparing the budding induction medium is as follows: 30 g / L sucrose and 6.5 g / L agar are added to MS medium to obtain the basic medium, and then 4 mg / L 6BA, 1 mg / L NAA and 1 mg / L (TDZ) are added. Finally, the pH of the medium is adjusted to 5.8.
[0034] 5) Seal the inoculated culture flasks with the caps and place them in an environment of 27℃, light intensity of 1250 lx, and light duration of 16 hours / day for 15 days.
[0035] 6) Screen the optimal bud induction medium. Add 30 g / L sucrose and 6.5 g / L agar to MS medium to obtain the basal medium. Then add plant growth regulators 6BA, NAA and TDZ (set TDZ concentration from 0 to 2.5 mg / L, with a concentration gradient of 0.5 mg / L, add 1.0 mg / L 6BA and 0.5 mg / L NAA to prepare 5 concentration gradients, numbered A, B, C, D and E). Adjust the pH of the medium to 5.8. Take the samples from step 5) and inoculate 20 bottles of each medium combination, inoculating one bud in each bottle. Culture for 30 days under the conditions of temperature 27℃±1℃, light intensity 1000lx-1500lx, and light duration 16 hours / day. Determine the optimal medium based on the overall condition of the bud cluster.
[0036] 7) Based on the screening results in step 6), further screen the concentration of plant growth regulators: Set the concentration of 6BA to 0.5-2.5 mg / L, with a concentration gradient of 0.5 mg / L, add 0.5 mg / L NAA to prepare 5 concentration gradients, numbered F, A, G, H, and I. Adjust the pH of the culture medium to 5.8. Take the samples from step 5), inoculate 20 bottles of each culture medium combination, inoculate one bud in each bottle, and culture for 30 days under the conditions of temperature 27℃, light intensity 1250 lx-, and light duration 16 hours / day; finally determine the optimal culture medium for inducing clustered buds based on the overall state of the clustered buds.
[0037] 8) Take the clustered buds and divide them into individual buds. Transfer them into rooting medium and culture them at a temperature of 27℃, a light intensity of 1250lx, and a light duration of 16 hours / day for 30 days.
[0038] The method for preparing the rooting induction medium is as follows: 30 g / L sucrose and 6.5 g / L agar are added to 1 / 2 MS medium to obtain the basal medium, then 3 mg / L NAA is added, and finally the pH of the medium is adjusted to 5.8.
[0039] 11) Statistical analysis of rooting rate and rooting status.
[0040] The following are the experimental results.
[0041] (1) The screening and results of the optimal bud induction medium were as follows: The concentration range of thiabendazole (TDZ) was set at 0-2.5 mg / L, and 1.0 mg / L 6-benzylaminopurine (6BA) and 0.5 mg / L naphthaleneacetic acid (NAA) were added to prepare 5 concentration gradients, numbered A, B, C, D, and E; the pH of the medium was adjusted to 5.8, and 20 bottles were inoculated for each medium combination and cultured for 30 days; the specific medium formulation is shown in Table 1, and the bud induction status is shown in Table 2. Figure 1 From Table 1 and Figure 1 It can be seen that: While maintaining constant concentrations of 1.0 mg / L 6-BA and 0.5 mg / L NAA, changing the TDZ concentration after 30 days of cultivation resulted in a decrease in the number of shoot clusters as the TDZ concentration increased. The highest number of shoots was observed with no TDZ addition, averaging 20, while the lowest number was observed with a TDZ concentration of 2.5 mg / L, averaging 9. Increased TDZ concentration led to reddish and abnormally curled leaves in the shoot clusters, and a decrease in overall plant vigor. (See Table 1 and...) Figure 1 It can be seen that the culture medium labeled A has the best overall condition for inducing clustered shoots.
[0042] Table 1. Effects of different TDZ concentrations on the induction of shoot clusters in Ixora coccinea
[0043]
[0044]
[0045] (2) Based on culture medium A, 6-benzylaminopurine (6BA) concentration ranged from 0.5-2.5 mg / L, and 0.5 mg / L naphthaleneacetic acid (NAA) was added to prepare 5 concentration gradients, numbered F, A, G, H, and I; the pH of the culture medium was adjusted to 5.8, and 20 bottles were inoculated for each culture medium combination. Cultured for 30 days, the specific culture medium formulations are shown in Table 2, and the induction status of shoot clusters is shown in [Table 2]. Figure 2 From Table 2 and Figure 2It can be seen that, while keeping the NAA concentration constant at 0.5 mg / L, changing the 6BA concentration and culturing for 30 days resulted in a decrease in the number of shoot clusters as the 6BA concentration increased. The highest number of shoots was observed at 0.5 mg / L and 1.0 mg / L, averaging 20, while the lowest number was observed at 2.5 mg / L, averaging 8. At 6BA concentrations above 1.5 mg / L, the leaves of the shoot clusters gradually turned red and became deformed and curled, the overall vigor of the plants decreased, and a small number of shoots died. The shoot clusters at a 6BA concentration of 0.5 mg / L were more vigorous and robust than those in the 1.0 mg / L group. These results indicate that the culture medium labeled F produced the best overall condition for the shoot clusters.
[0046] Table 2. Effects of different 6BA concentrations in culture medium on the induction of shoot clusters in Ixora coccinea.
[0047]
[0048] (3) Transfer the clustered shoots induced by F medium to rooting medium, inoculate 30 bottles, one shoot per bottle, and culture them under conditions of 27℃±1℃, light intensity of 1000lx-1500lx, and light duration of 16 hours / day for 30 days; after 30 days, the overall rooting rate reached 80%, and the root system was well-developed and robust with many fibrous roots, such as Figure 3 .
[0049] Therefore, in summary, MS + 30 g / L sucrose + 6.5 g / L agar + 0.5 mg / L 6-BA + 0.5 mg / L NAA is the most suitable culture medium for inducing clustered shoots in Ixora chinensis.
[0050] Example 2
[0051] A method for rapid propagation of Ixora includes the following steps:
[0052] 1) On a clear morning at 9 a.m., select a healthy, semi-lignified Ixora branch with terminal buds and mature leaves from the current year as an explant, and cut the entire branch off with pruning shears.
[0053] 2) After pruning the leaves of the Ixora chinensis branches, leave 3-4 cm below the terminal bud and place them in a 500 mL culture bottle with 4-5 branches per bottle. On a clean bench sterilized with ultraviolet light, first soak the explants in 75% ethanol for 32 seconds, then rinse them once with sterile water. Add 2% sodium hypochlorite solution to cover the explants, then add 1‰-2‰ Tween 80 and sonicate at 500W in the dark for 8 minutes to ensure thorough sterilization. Finally, rinse them twice with sterile water.
[0054] 3) Transfer the culture flask to a sterile laminar flow hood, fill it with 2% sodium hypochlorite solution, and soak it in the dark for 5 minutes. During the soaking process, gently shake the Erlenmeyer flask to ensure thorough sterilization. Finally, rinse it 3 times with sterile water.
[0055] 4) Place the branches on sterile filter paper and let them dry completely. Then, use sterile forceps and a scalpel to completely remove the oxidized and blackened parts of the branches. Make a slanted cut at the lower morphological end of the branches. During the inoculation process, inoculate the branches at a 45° angle into the bud induction medium.
[0056] The method for preparing the budding induction medium is as follows: 30 g / L sucrose and 6.5 g / L agar are added to MS medium to obtain the basic medium, and then 4 mg / L 6-BA, 1 mg / L NAA and 1 mg / L TDZ are added. Finally, the pH of the medium is adjusted to 5.8.
[0057] 5) After sealing the inoculated culture flasks, place them in an environment with a temperature of 26℃, a light intensity of 1000 lx, and a light duration of 16 hours / day for 15 days.
[0058] 6) After sterilizing the culture bottle with 75% alcohol, place it on a sterile operating table. Use sterile forceps to pick up the branch and place it on sterile filter paper. Use a sterile scalpel to cut off the tender new terminal bud of the branch and make an oblique cut at its morphological lower end. Inoculate the branch at a 45° angle into the cluster bud induction medium.
[0059] The method for preparing the bud induction medium is as follows: 30 g / L sucrose and 6.5 g / L agar are added to MS medium to obtain the basal medium, then 0.5 mg / L 6-BA and 0.5 mg / L NAA are added, and finally the pH of the medium is adjusted to 5.8.
[0060] 7) After sealing the inoculated culture flasks, place them in an environment with a temperature of 27℃±1℃, a light intensity of 1000lx, and a light duration of 16 hours / day for 30 days.
[0061] 8) Take the clustered buds and divide them into individual buds. Transfer them into rooting medium and culture them under the conditions of 28℃, 1000lx light intensity, and 16 hours / day for 30 days. Calculate the rooting rate.
[0062] The rooting induction medium is prepared by adding 30 g / L sucrose and 6.5 g / L agar to 1 / 2 MS medium to obtain the basal medium, then adding 3 mg / L NAA, and finally adjusting the pH of the medium to 5.8.
[0063] Example 3
[0064] A method for rapid propagation of Ixora includes the following steps:
[0065] 1) On a clear morning at 10 a.m., select a healthy, semi-lignified Ixora branch with terminal buds and mature leaves from the current year as an explant, and cut the entire branch off with pruning shears.
[0066] 2) After pruning the leaves of the Ixora chinensis branches, leave 3-4 cm below the terminal bud and place them in a 500 mL culture bottle with 4-5 branches per bottle. On a clean bench sterilized with ultraviolet light, first soak the explants in 75% ethanol for 30±2 seconds, then rinse them once with sterile water. Add 2% sodium hypochlorite solution to cover the explants, then add 1‰-2‰ Tween 80, and sonicate at 500W in the dark for 10 minutes to ensure thorough sterilization. Finally, rinse them three times with sterile water.
[0067] 3) Transfer the culture flask to a sterile laminar flow hood, fill it with 2% sodium hypochlorite solution, and soak it in the dark for 8 minutes. Gently shake the Erlenmeyer flask during the soaking process to ensure thorough sterilization. Finally, rinse it twice with sterile water.
[0068] 4) Place the branches on sterile filter paper and let them dry completely. Then, use sterile forceps and a scalpel to completely remove the oxidized and blackened parts of the branches. Make a slanted cut at the lower morphological end of the branches. During the inoculation process, inoculate the branches at a 45° angle into the bud induction medium.
[0069] The method for preparing the budding induction medium is as follows: 30 g / L sucrose and 6.5 g / L agar are added to MS medium to obtain the basic medium, and then 4 mg / L 6-6-BA, 1 mg / L NAA and 1 mg / L LTDZ are added. Finally, the pH of the medium is adjusted to 5.8.
[0070] 5) After sealing the inoculated culture flasks, place them in an environment with a temperature of 26℃, a light intensity of 1500 lx, and a light duration of 16 hours / day for 15 days.
[0071] 6) After sterilizing the culture bottle with 75% alcohol, place it on a sterile operating table. Use sterile forceps to pick up the branch and place it on sterile filter paper. Use a sterile scalpel to cut off the tender new terminal bud of the branch and make an oblique cut at its morphological lower end. Inoculate the branch at a 45° angle into the cluster bud induction medium.
[0072] The method for preparing the bud induction medium is as follows: 30 g / L sucrose and 6.5 g / L agar are added to MS medium to obtain the basal medium, then 0.5 mg / L 6-BA and 0.5 mg / L NAA are added, and finally the pH of the medium is adjusted to 5.8.
[0073] 7) After sealing the inoculated culture flasks, place them in an environment with a temperature of 27℃, a light intensity of 1500 lx, and a light duration of 16 hours / day for 30 days.
[0074] 8) Take the clustered buds and divide them into individual buds. Transfer them into rooting medium and culture them under the conditions of 27℃, light intensity of 1500lx and light time of 16 hours / day for 30 days. Calculate the rooting rate.
[0075] The rooting induction medium is prepared by adding 30 g / L sucrose and 6.5 g / L agar to 1 / 2 MS medium to obtain the basal medium, then adding 3 mg / L NAA, and finally adjusting the pH of the medium to 5.8.
[0076] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.
Claims
1. A method for rapid propagation of Ixora chinensis, characterized in that, Includes the following steps: 1) Select healthy, semi-lignified Ixora branches with terminal buds and mature leaves from the current year as explants; 2) After pruning the leaves of the Ixora branches, place them in a culture bottle with 4-5 branches per bottle, leaving 3-4 cm below the terminal bud. Disinfect the branches. 3) Place the branches on sterile filter paper and let them dry completely. Then, use sterile forceps and a scalpel to completely remove the oxidized and blackened parts of the branches. Make a slanted cut at the lower morphological end of the branches. During the inoculation process, inoculate the branches at a 45° angle into the bud induction medium. The method for preparing the budding induction medium is as follows: 30 g / L sucrose and 6.5 g / L agar are added to MS medium to obtain the basic medium, and then 4 mg / L 6-BA, 1 mg / L NAA and 1 mg / L TDZ are added. Finally, the pH of the medium is adjusted to 5.
8. 4) After sealing the inoculated culture bottles, place them in an environment with a temperature of 27℃±1℃, a light intensity of 1000lx-1500lx, and a light duration of 16 hours / day for 15 days. 5) After sterilizing the culture bottle, place it on a sterile operating table, use sterile forceps to pick up the branch and place it on sterile filter paper, use a sterile scalpel to cut off the tender new terminal bud of the branch, and make an oblique cut at the lower morphological end. Inoculate the branch at a 45° angle into the cluster bud induction medium. The method for preparing the bud induction medium is as follows: 30 g / L sucrose and 6.5 g / L agar are added to MS medium to obtain the basal medium, then 0.5 mg / L 6-BA and 0.5 mg / L NAA are added, and finally the pH of the medium is adjusted to 5.
8. 6) After sealing the inoculated culture flasks, place them in an environment with a temperature of 27℃±1℃, a light intensity of 1000lx-1500lx, and a light duration of 16 hours / day for 30 days. 7) Take the clustered buds and divide them into individual buds. Transfer them into rooting medium and culture them under the conditions of temperature 27℃±1℃, light intensity 1000lx-1500lx, and light time 16 hours / day for 30 days. The rooting induction medium was prepared by adding 30 g / L sucrose and 6.5 g / L agar to 1 / 2 MS medium to obtain the basal medium, then adding 3 mg / L NAA, and finally adjusting the pH of the medium to 5.
8.
2. The method for rapid propagation of Ixora chinensis according to claim 1, characterized in that, In step 1), the explant is collected between 9:00 and 11:00 AM on a sunny morning.
3. The method for rapid propagation of Ixora chinensis according to claim 1, characterized in that, In step 1), the branch disinfection process described in step 2) is as follows: Transfer the culture bottle to a sterile laminar flow hood, soak it in 75% ethanol for 30±2s, then rinse it once with sterile water; add 2% sodium hypochlorite solution to cover the explant, then add 1‰-2‰ Tween 80, sonicate at 500W in the dark for 8-10 minutes, and finally rinse it 2-3 times with sterile water; transfer the culture bottle to the sterile laminar flow hood again, fill it with 2% sodium hypochlorite solution, soak it in the dark for 5-8 minutes, gently agitate the culture bottle during the soaking process to ensure thorough disinfection, and finally rinse it 2-3 times with sterile water.