Open type cold region Chinese rose tissue culture seedling propagation method
By using chlorometa-xylenol and miconazole nitrate as antibacterial agents, combined with open culture media and simplified operating procedures, the environmental and equipment limitations of cutting propagation of cold-resistant roses were overcome, achieving efficient tissue culture seedling propagation and improving the plant regeneration rate and growth status of cold-resistant roses.
Patent Information
- Application Number
- CN202511074858.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-01
- Publication Date
- 2025-11-11
AI Technical Summary
The propagation of cold-climate roses by cuttings is limited by the environment and equipment. In the existing open tissue culture technology, antibacterial agents cause severe browning and vitrification, resulting in low plant regeneration rate and failing to meet the needs of large-scale propagation.
Using chloro-meta-xylenol and miconazole nitrate as antibacterial agents, combined with open culture media and simplified operating procedures, including explant treatment, culture medium preparation and tissue culture seedling transplantation, avoids a strictly sterile environment and reduces equipment costs.
With low-cost investment, rapid propagation of tissue culture seedlings of cold-region roses has been achieved, improving the induction rate and rooting rate of clustered buds, reducing the pollution rate and browning rate, and enhancing the plant growth status.
Smart Images
Figure CN120918100A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of tissue culture seedling propagation technology, specifically relating to an open-type cold-region rose tissue culture seedling propagation method. Background Technology
[0003] For a long time, the main propagation method for cold-climate roses has been cutting propagation, primarily due to its low cost and relatively simple operation. However, cutting propagation is limited by many factors such as the timing of cuttings, temperature, humidity, and light, making it impossible to rapidly and massively propagate many cold-climate rose varieties through this method. This, in turn, restricts the application and promotion of many excellent cold-climate rose varieties in the market.
[0004] Tissue culture technology offers advantages such as being unrestricted by region or season, having a short seedling acquisition cycle, and high propagation rate. It not only preserves the superior traits of the parent plant but also facilitates industrialized production. Therefore, the use of plant tissue culture technology for rose seedling propagation has been widely adopted. However, because rose seedling propagation using plant tissue culture technology relies on sterile facilities and environments, the production process requires significant capital investment in factory construction and the purchase of ultra-clean equipment, resulting in substantial costs and thus limiting the application of this technology.
[0005] Open tissue culture technology for plants refers to the use of antibacterial agents to inhibit the reproduction of bacteria and fungi on the culture medium, allowing plant tissue culture to be conducted outside the strictly sterile environment. This eliminates the limitations of high-temperature, high-pressure sterilizers and laminar flow hoods, enabling plant tissue culture in an open, natural environment. Compared to conventional tissue culture, open tissue culture removes the need for a strictly sterile environment, reducing the requirements for equipment and environment, simplifying the process, and significantly reducing seedling costs. However, most antibacterial agents used in open tissue culture can harm plants, causing browning or vitrification of plant tissues during culture. Therefore, different plants require different types of antibacterial agents. Currently, open tissue culture technology has been applied to the tissue culture of potatoes, sugarcane, azaleas, and plum blossoms, but it has not been reported in the tissue culture of roses. Furthermore, the existing reported antibacterial agents are not suitable for cold-hardy roses, resulting in significant browning and vitrification during open culture, leading to extremely low plant regeneration rates and failing to meet production needs. Summary of the Invention
[0006] The purpose of this invention is to provide an open-type tissue culture propagation method for cold-resistant roses to reduce browning and vitrification during open cultivation and improve plant regeneration rate.
[0007] An open-type cold-region rose tissue culture propagation method includes:
[0008] 1) Induction culture
[0009] a. Pre-treatment of explants: Mix mother solution A and mother solution B at a volume ratio of 1:1, dilute 20 times, and spray semi-lignified branches of cold-resistant roses once a day for three days;
[0010] b. Induction culture: Take semi-lignified branches of cold-region roses, rinse them with running water, cut them into 2cm segments containing axillary buds as explants, soak them in 75% alcohol for 30s, then mix mother solution A and mother solution B at a volume ratio of 1:1, dilute them 100 times, soak them for another 30min, take them out, dry them, and inoculate them into tissue culture bottles in the natural growth direction for induction culture.
[0011] c. Open rooting culture: After the obtained clustered shoots are divided, they are inoculated into open rooting culture medium according to their growth direction;
[0012] d. Transplanting of tissue culture seedlings: After rooting, transplant the cold-resistant rose tissue culture seedlings into a transplanting substrate with a weight ratio of coconut coir, vermiculite and perlite of 5:4:1. After transplanting, mix mother solution A and mother solution B at a volume ratio of 1:1, dilute 200 times, thoroughly water the transplanting substrate, cover with a small arched greenhouse, and cover with film to retain moisture.
[0013] The culture medium for induction culture is as follows: 30g of sucrose and 5.5g of agar are added to each liter of MS medium, and the mixture is heated to boiling to fully dissolve the agar. When the temperature of the culture medium drops to 60-70℃, the following concentrations are added to the culture medium: 1.0-2.0mg / L of 6-BA, 0.5-1.5mg / L of IBA, 10ml / L of antibacterial agent stock solution A, and 10ml / L of antibacterial agent stock solution B.
[0014] The rooting culture medium is as follows: 30g of sucrose and 5.5g of agar are added to each liter of MS medium, and the mixture is heated to boiling to fully dissolve the agar. When the temperature of the medium drops to 60-70℃, 0.5 mg / L of IAA, 10 ml / L of antibacterial agent stock solution A, and 10 ml / L of antibacterial agent stock solution B are added to the medium.
[0015] Before the explants are inoculated into the tissue culture flasks, 3 mm is cut off from both the top and bottom ends.
[0016] This invention provides an open-type method for propagating cold-resistant rose tissue culture seedlings. Para-chloro-meta-xylenol (PCMX) and miconazole nitrate (MSDS) are used as antibacterial agents, applied respectively in various tissue culture stages including explant pretreatment, equipment sterilization, culture medium preparation, explant disinfection, and seedling transplantation. Explants are cut into 2cm segments, disinfected, and before being transferred to the tissue culture bottle, 3mm is cut off at both ends. This method frees the propagation process of cold-resistant rose tissue culture seedlings from environmental limitations. It achieves rapid propagation of rose tissue culture seedlings at a low cost (without requiring a strictly sterile environment, autoclave, or laminar flow hood).
[0017] 1) During induction culture, the induction rate of Hanjin No. 1 shoot clusters can reach 96.7%, which is higher than the 90.4% induction rate of conventional sterilization methods. At the same time, the contamination rate is reduced by 10%, the vitrification percentage is reduced by 10%, and the browning rate is reduced by 3.5%.
[0018] The 2021-EY-1 bud induction rate can reach 95.6%, which is higher than the 86.6% induction rate of conventional sterilization methods. At the same time, the contamination rate is reduced by 12.1%, the vitrification percentage is reduced by 22.5%, and the browning rate is reduced by 12.2%.
[0019] The induction rate of bud clusters in the climbing roses of Changbai Mountain can reach 98.6%, with a contamination rate of 5.2%, vitrification of 6.3%, and browning rate of 2.7%. The bud cluster induction rate of plants obtained by conventional sterilization methods is 93.3%, with a contamination rate of 14.3%, vitrification of 32.3%, and browning rate of 12.5%.
[0020] 2) During rooting culture: the rooting rate does not change significantly, but the growth is significantly improved compared with conventional culture, especially for Hanjin No. 1 and Changbai Mountain climbing roses. Attached Figure Description
[0021] Figure 1 The growth of the climbing rose from Changbai Mountain in an open culture medium and its flowering after transplanting;
[0022] Figure 2 Growth of 2021-EY-1 in open culture medium and flowering after transplanting;
[0023] Figure 3 The growth of Hanjin No. 1 in open culture medium and its flowering after transplanting;
[0024] Figure 4 Rooting status of tissue culture seedlings in a culture medium containing 10 ml / L antibacterial agent A and 10 ml / L antibacterial agent B;
[0025] Figure 5Rooting of tissue culture seedlings in a culture medium without added antibacterial agents. Detailed Implementation
[0026] Example 1: Open Breeding of Hanjin No. 1
[0027] 1. Pretreatment of explants: Semi-lignified stem segments of the cold-region rose variety "Hanjin No. 1" were used as explants. Three days before explant collection, the desired explant sites were sprayed with a mixture of stock solutions A and B diluted 20 times. The treated explant material was cut into small segments (2 cm each) containing buds and inoculated into the induction medium according to the growth direction. 30 bottles were inoculated for each treatment.
[0028] The mother liquor A is a 50 g / L para-chloro-meta-xylenol (PCMX) solution; the mother liquor B is a 20 g / L miconazole nitrate (MSDS).
[0029] 2. Prepare open induction medium: Using MS as the basal medium, add 30g of sucrose and 5.5g of agar per liter of medium, and heat to boiling to fully dissolve the agar. When the medium temperature drops to 60-70℃, add 1.5mg / L of 6-BA, 0.5mg / L of IBA, 10ml / L of antibacterial agent stock solution A, and 10ml / L of antibacterial agent stock solution B to the medium. Dispense into tissue culture bottles that have been treated with antibacterial agents, and seal the bottles for later use.
[0030] 3. Cut rose branches treated with antibacterial agent combination, remove hard thorns and leaves from the branches, cut into 2cm long stem segments with axillary buds in the middle, soak in 75% alcohol for 30 seconds, then soak in 100 times diluted stock solution A and B for 30 minutes, take out and let dry before use.
[0031] 4. Explant materials for culture media without added antibacterial agents were sterilized using conventional methods under a clean bench: Rose branches not treated with antibacterial agents were cut, removing thorns and leaves, and cut into 2cm long stem segments containing axillary buds. The segments were then immersed in 75% alcohol for 30 seconds in a clean bench, followed by treatment with 1% NaClO for 15 minutes, and rinsed 3-5 times with sterile water before use.
[0032] 5. Cut off 3mm from both ends of the stem segment and inoculate it into open induction medium containing different concentrations of antibacterial agent according to the growth direction. 30 bottles are inoculated for each group, and the inoculation is repeated three times.
[0033] 6. Investigate the contamination rate, vitrification percentage, browning rate, and induction rate after 40 days:
[0034] Contamination rate = (Number of contaminated explants / Total number of inoculated explants) * 100%;
[0035] Induction rate = (Number of explants producing clustered seedlings / Total number of inoculated explants) * 100%;
[0036] Browning rate = (Number of browned explants / Total number of inoculated explants) * 100%;
[0037] Vitrification percentage = (Number of vitrified explants / Total number of inoculated explants) * 100%.
[0038] 7. Experimental results showed that the induction rate of shoot clusters was highest at 96.7% when the concentrations of antibacterial agent A and antibacterial agent B in the culture medium were 10 ml / L and 10 ml / L, respectively. This was higher than the 90.4% induction rate achieved by conventional sterilization methods. Simultaneously, the contamination rate decreased by 10%, the vitrification percentage decreased by 10%, and the browning rate decreased by 3.5%. All these values were lower than those obtained by conventional sterilization methods, indicating good plant growth.
[0039] .
[0040] Example 2: Open Breeding of 2021-EY-1
[0041] 1. Pretreatment of explants: Semi-lignified stem segments of the Russian-imported hardy rose 2021-EY-1 were used as explants. Three days before explant collection, the desired explant sites were sprayed with a mixture of stock solutions A and B diluted 20 times. The treated explant material was cut into small segments (2 cm each) containing buds and inoculated into the induction medium in the direction of growth. 30 bottles were inoculated for each treatment.
[0042] 2. Prepare open induction medium: Using MS as the basal medium, add 30g of sucrose and 5.5g of agar per liter of medium, and heat to boiling to fully dissolve the agar. When the medium temperature drops to 60-70℃, add 2.0mg / L 6-BA, 0.5mg / L IAA, 10ml / L of antibacterial stock solution A, and 10ml / L of antibacterial stock solution B to the medium. Dispense into tissue culture flasks that have been treated with the antibacterial agents, and seal the flasks for later use.
[0043] 3. Cut rose branches treated with antibacterial agent combination, remove hard thorns and leaves from the branches, cut into 2cm long stem segments with axillary buds in the middle, soak in 75% alcohol for 30 seconds, then soak in 100 times diluted stock solution A and B for 30 minutes, take out and let dry before use.
[0044] 4. Explant materials for culture media without added antibacterial agents were sterilized using conventional methods under a clean bench: Rose branches not treated with antibacterial agents were cut, removing thorns and leaves, and cut into 2cm long stem segments containing axillary buds. The segments were then immersed in 75% alcohol for 30 seconds in a clean bench, followed by treatment with 1% NaClO for 15 minutes, and rinsed 3-5 times with sterile water before use.
[0045] 5. Cut off 3mm from both ends of the stem segment and inoculate it into open induction medium containing different concentrations of antibacterial agent according to the growth direction. 30 bottles are inoculated for each group, and the inoculation is repeated three times.
[0046] 6. Investigate the contamination rate, vitrification percentage, browning rate, and induction rate after 40 days:
[0047] Contamination rate = (Number of contaminated explants / Total number of inoculated explants) * 100%;
[0048] Induction rate = (Number of explants producing clustered seedlings / Total number of inoculated explants) * 100%;
[0049] Browning rate = (Number of browned explants / Total number of inoculated explants) * 100%;
[0050] Vitrification percentage = (Number of vitrified explants / Total number of inoculated explants) * 100%.
[0051] 7. Experimental results showed that the highest induction rate of shoot clusters (95.6%) was achieved when the concentrations of antibacterial agent A and antibacterial agent B in the culture medium were 10 ml / L and 10 ml / L, respectively. This was higher than the 86.6% induction rate obtained by conventional sterilization methods. Simultaneously, the contamination rate decreased by 12.1%, the vitrification percentage decreased by 22.5%, and the browning rate decreased by 12.2%. All these values were lower than those obtained by conventional sterilization methods, indicating good plant growth.
[0052] .
[0053] Example 3: Open Propagation of Climbing Roses from Changbai Mountain
[0054] 1. Pretreatment of explants: Semi-lignified stem segments of the climbing rose from Changbai Mountain were used as explants. Three days before explant collection, the desired explant sites were sprayed with a mixture of stock solutions A and B diluted 20 times. The treated explant material was cut into small segments (2 cm each) containing buds and inoculated into the induction medium in the direction of growth. 30 bottles were inoculated for each treatment.
[0055] 2. Prepare open induction medium: Using MS as the basal medium, add 30g of sucrose and 5.5g of agar per liter of medium, and heat to boiling to fully dissolve the agar. When the medium temperature drops to 60-70℃, add 1.0mg / L of 6-BA, 0.4mg / L of NAA, 10ml / L of antibacterial agent stock solution A, and 10ml / L of antibacterial agent stock solution B to the medium. Dispense into tissue culture bottles that have been treated with antibacterial agents, and seal the bottles for later use.
[0056] 3. Cut rose branches treated with antibacterial agent combination, remove hard thorns and leaves from the branches, cut into 2cm long stem segments with axillary buds in the middle, soak in 75% alcohol for 30 seconds, then soak in 100 times diluted stock solution A and B for 30 minutes, take out and let dry before use.
[0057] 4. Explant materials for culture media without added antibacterial agents were sterilized using conventional methods under a clean bench: Rose branches not treated with antibacterial agents were cut, removing thorns and leaves, and cut into 2cm long stem segments containing axillary buds. The segments were then immersed in 75% alcohol for 30 seconds in a clean bench, followed by treatment with 1% NaClO for 15 minutes, and rinsed 3-5 times with sterile water before use.
[0058] 5. Cut off 3mm from both ends of the stem segment and inoculate it into open induction medium containing different concentrations of antibacterial agent according to the growth direction. 30 bottles are inoculated for each group, and the inoculation is repeated three times.
[0059] 6. Investigate the contamination rate, vitrification percentage, browning rate, and induction rate after 40 days:
[0060] Contamination rate = (Number of contaminated explants / Total number of inoculated explants) * 100%;
[0061] Induction rate = (Number of explants producing clustered seedlings / Total number of inoculated explants) * 100%;
[0062] Browning rate = (Number of browned explants / Total number of inoculated explants) * 100%;
[0063] Vitrification percentage = (Number of vitrified explants / Total number of inoculated explants) * 100%.
[0064] 7. Experimental results showed that when the concentration of antibacterial agent A in the culture medium was 10 ml / L and the concentration of antibacterial agent B was 10 ml / L, the induction rate of clustered shoots could reach the maximum of 98.6%, the contamination rate was 5.2%, the vitrification rate was 6.3%, and the browning rate was 2.7%. The induction rate of clustered shoots of plants obtained by conventional sterilization methods was 93.3%, the contamination rate was 14.3%, the vitrification rate was 32.3%, and the browning rate was 12.5%.
[0065]
[0066] Example 4: Determination of the optimal open rooting medium for Hanjin No. 1, 2021-EY-1 and Changbai Mountain climbing roses
[0067] 1. Prepare open rooting medium: Using MS as the basal medium, add 30g of sucrose and 5.5g of agar per liter of medium, heat to boiling, and ensure the agar is fully dissolved. When the medium temperature drops to 60-70℃, add 0.5 mg / L IAA, 5 ml / L-20 ml / L of antibacterial agent stock solution A, and 5 ml / L-20 ml / L of antibacterial agent stock solution B to the medium. Dispense into tissue culture flasks that have been treated with the antibacterial agents, and seal the flasks for later use.
[0068] 2. After dividing the obtained clustered shoots, inoculate them separately according to their growth direction into open rooting medium containing different concentrations of antibacterial agents.
[0069] 3. Investigate rooting rate and growth after 40 days:
[0070] Rooting rate = Number of rooted tissue culture seedlings / Total number of inoculated tissue culture seedlings * 100%.
[0071] 4. Experimental results showed that, comparing the results of rooting induction experiments for different varieties, it was found that for the same variety, the rooting rate and growth of tissue culture seedlings were improved in the culture medium supplemented with 10 ml / L of antibacterial agent A and 10 ml / L of antibacterial agent B, reaching a maximum of 100%. As the concentration of antibacterial agents in the culture medium increased, although the rooting rate of tissue culture seedlings did not decrease significantly, root development was significantly slower and deformed roots appeared, and the growth of tissue culture seedlings gradually weakened. Therefore, this culture medium can be determined as the optimal open rooting medium.
[0072]
[0073] Example 5: Transplanting of tissue-cultured seedlings of Hanjin No. 1, 2021-EY-1 and Changbai Mountain climbing roses.
[0074] 1. After rooting, transplant the cold-resistant rose tissue culture seedlings into a transplanting substrate of coconut coir:vermiculite:perlite = 5:4:1. After transplanting, thoroughly water with stock solutions A and B diluted 200 times (for the control, water thoroughly with clean water), cover with a small arched greenhouse, and cover with film to retain moisture.
[0075] 2.15 days later, the survival rate of the tissue culture seedlings was investigated: Survival rate = Number of surviving tissue culture seedlings / Total number of transplanted tissue culture seedlings * 100%.
[0076] 3. Experimental results show that after treatment with mother liquor A and B diluted 200 times, the survival rate can reach 96%, which is 23% higher than that of water.
Claims
1. An open-type cold-region rose tissue culture propagation method, comprising: 1) Induction culture a. Pre-treatment of explants: Mix mother solution A and mother solution B at a volume ratio of 1:1, dilute 20 times, and spray semi-lignified branches of cold-resistant roses once a day for three days; b. Induction culture: Take semi-lignified branches of cold-region rose, rinse them with running water, cut them into 2cm segments containing axillary buds as explants, soak them in 75% alcohol for 30s, then mix mother solution A and mother solution B at a volume ratio of 1:1, dilute 100 times, soak for another 30min, take them out, dry them, and inoculate them into tissue culture bottles in the natural growth direction for induction culture. The mother liquor A is a 50 g / L solution of p-chloro-meta-xylenol; the mother liquor B is a 20 g / L solution of miconazole nitrate. c. Open rooting culture: After the obtained clustered shoots are divided, they are inoculated into open rooting culture medium according to their growth direction; d. Transplanting tissue culture seedlings After rooting, the tissue culture seedlings of cold-resistant roses were transplanted into a transplanting substrate consisting of coconut coir, vermiculite, and perlite in a weight ratio of 5:4:
1. After transplanting, the mother solution A and mother solution B were mixed in a volume ratio of 1:1 and diluted 200 times. The transplanting substrate was then thoroughly watered, and a small arched greenhouse was placed over it to retain moisture. The culture medium for induction culture is as follows: 30g of sucrose and 5.5g of agar are added to each liter of MS medium, and the mixture is heated to boiling to fully dissolve the agar. When the temperature of the culture medium drops to 60-70℃, the following concentrations are added to the culture medium: 1.0-2.0mg / L of 6-BA, 0.5-1.5mg / L of IBA, 10ml / L of antibacterial agent stock solution A, and 10ml / L of antibacterial agent stock solution B. The rooting culture medium is as follows: 30g of sucrose and 5.5g of agar are added to each liter of MS medium, and the mixture is heated to boiling to fully dissolve the agar. When the temperature of the medium drops to 60-70℃, 0.5 mg / L of IAA, 10 ml / L of antibacterial agent stock solution A, and 10 ml / L of antibacterial agent stock solution B are added to the medium.
2. The method for propagating cold-resistant rose tissue culture seedlings according to claim 1, characterized in that: Before the explants are inoculated into the tissue culture flasks, 3 mm is cut off from both the top and bottom ends.