Tissue culture and rapid propagation method of salvia pseudocerasus
By using a specific formula for inducing new shoots and rooting culture medium, as well as sterilization methods, rapid propagation of cherry sage was successfully achieved, solving the problem of depletion of cherry sage germplasm resources and meeting industrial needs.
Patent Information
- Application Number
- CN202411446530.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-16
- Publication Date
- 2025-01-28
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The lack of existing technologies for rapid propagation of cherry sage through tissue culture has led to increased demand for raw materials and depletion of germplasm resources.
New shoot induction and rooting media with specific formulations, including MS + 0.1 mg/L NAA + 0.1-2 mg/L BA + 30 g/L sucrose + 7 g/L agar and 1/2 MS or MS + 0.2 mg/L NAA + 0.05-0.5 mg/L paclobutrazol + 30 g/L sucrose + 7 g/L agar, combined with sterilization methods, were used to induce new shoots and promote rooting, thus obtaining sterile cherry sage seedlings.
It has enabled the rapid propagation of cherry sage, with a high rooting rate, well-developed root system, and moderate seedling height, solving the problem of depletion of cherry sage germplasm resources and meeting industrial needs.
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Figure CN119366443A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of plant rapid propagation and relates to a tissue culture rapid propagation method of cherry sage. Background Art
[0002] Cherry sage (Salvia greggii) is a perennial herbaceous plant of the genus Salvia in the family Lamiaceae, native to Texas to Mexico. The leaves are single and opposite, and are ovate or elliptical with round teeth or round serrations on the edges; the inflorescence is racemose, and the corolla is lip-shaped, with peach red, dark red, pink, apricot yellow, white, etc.; the upper lobes of the upper lip are velvety, the lower lip is three-lobed, the middle lobe is larger, concave or two-lobed, and the lower lobe is wide and drooping, cracked on both sides, and concave at the end; it is named because it exudes a faint cherry fragrance. This species has beautiful leaves, dark green leaves, and pink corolla, and is also called "Beauty Cherry". The flowering period of cherry sage is very long, and it can bloom from May to November in the Yangtze River Delta region. Cherry sage is resistant to high temperatures but not low temperatures. The minimum maintenance temperature in winter should be kept above 7 degrees, otherwise it will be frostbitten. The characteristics of this plant enable it to remain green all year round, with fragrant leaves and flowers, and high ornamental value.
[0003] Cherry sage is an important aromatic herb that adds a pleasant flavor when used as a seasoning in cooking and has good antibacterial and anti-toxic effects. Its flowers can also be used to make tea, and its fragrance is strong and long-lasting. It can also remove body fat, promote blood circulation, and have a beauty and skin care effect. At the same time, carnosyl phenol has anti-cancer effects, and sage extract has the effect of treating rheumatism.
[0004] Sage has become a focus of food research to cope with the growing health awareness and the growing demand for healthy and nutritious food. It is expected to be used in the food and pharmaceutical fields. In recent years, the demand for sage has gradually increased, and its development has been getting better and better, which has led to a gradual increase in the demand for raw materials. Due to the increase in industrial demand and the depletion of a large number of wild populations, sage germplasm resources are on the verge of exhaustion. Tissue culture can obtain a large number of high-quality seedlings in a short period of time and provide a theoretical basis for the physiological, biochemical and genetic transformation of sage. At present, there are some reports on tissue culture of Salvia plants, such as obtaining regenerated plants of Salvia farinacea through apical bud culture; obtaining regenerated plants of Salvia przewalskii through leaves; there are also some reports on the tissue culture of Salvia sphenanthera abroad, such as the first micro-propagation study of Salvia sphenanthera using three explants: young leaves, cotyledons and stem segments with buds; and establishing a tissue culture system of Salvia sphenanthera using stem segments as materials.
[0005] However, there are no reports on the rapid propagation technology of cherry sage through tissue culture. Summary of the invention
[0006] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a tissue culture and rapid propagation method of cherry sage, which is used for the rapid propagation of cherry sage.
[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:
[0008] A tissue culture rapid propagation method for cherry sage comprises the following steps: sterilizing and disinfecting cherry sage tender branches and then inoculating them into a new bud induction medium to induce new buds; and then transferring them to a rooting medium for subculture to obtain sterile cherry sage seedlings.
[0009] The formula of the new sprout induction medium is: MS+0.1 mg / L NAA+0.1-2 mg / L BA+30 g / L sucrose+7 g / L agar.
[0010] The formula of the new sprout induction medium is: MS+0.1 mg / L NAA+2 mg / L BA+30 g / L sucrose+7 g / L agar.
[0011] The formula of the rooting medium is: 1 / 2MS or MS+0.2mg / L NAA+0.05-0.5mg / L uniconazole+30g / L sucrose+7g / L agar.
[0012] The formula of the rooting medium is: 1 / 2MS or MS+0.2mg / L NAA+0.2mg / L uniconazole+30g / L sucrose+7g / L agar.
[0013] The formula of the rooting medium is: 1 / 2MS+0.2mg / L NAA+0.2mg / L uniconazole+30g / L sucrose+7g / L agar.
[0014] The sterilization method comprises the following steps: sterilizing the cherry sage twigs with a 70% ethanol solution for 0.5 minutes, then soaking them in 0.1% mercuric chloride for 5 minutes, and then washing them with sterile water for 3-4 times.
[0015] The tissue culture and rapid propagation method of cherry sage includes the following specific steps:
[0016] 1) Transplant the cherry sage plants grown outdoors into pots and place them in the laboratory for one month. When new shoots emerge from the old shoots, cut off the new shoots for tissue culture;
[0017] 2) Sterilize the cherry sage shoots with 70% ethanol solution for 0.5 min, then soak them in 0.1% mercuric chloride for 5 min, and rinse them with sterile water 3-4 times after soaking;
[0018] 3) After rinsing, the shoots are moved to a clean workbench and placed on a culture dish covered with sterile paper for cutting. The shoots are cut into stem segments with 1-2 leaves, inoculated into a new shoot induction medium, and placed in a culture room with a temperature of 25-28° C., a light intensity of 1500-3000 Lux, and a 12-hour light and 12-hour dark photoperiod for new shoot induction culture;
[0019] 4) After new shoots grow out of the stem segments, they are inoculated into a rooting medium and placed in a culture room with a temperature of 25-28° C., a light intensity of 1500-3000 Lux, and a photoperiod of 12 h light and 12 h dark for rooting culture, thereby finally obtaining complete sterile cherry sage seedlings.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] 1) The cherry sage seedlings of the present invention take root 100% in a new sprout induction medium without cytokinin BA; in a new sprout induction medium with a lower concentration of cytokinin BA, new roots still grow; as the cytokinin concentration increases, the rooting rate becomes lower and lower until the rooting rate is zero when the BA concentration is 2 mg / L. The results show that BA in the new sprout induction medium has the effect of inhibiting rooting.
[0022] 2) The results of the present invention show that with the increase of BA concentration, the new sprout induction rate gradually increases. When the BA concentration is 2 mg / L, the new sprout induction rate is the highest, which is 100%.
[0023] 3) The results of the present invention show that on the 1 / 2MS + NAA 0.2mg / L + 0.5mg / L clofosinate medium, the seedling height is the shortest, which is 7.7cm; with the increase of the clofosinate concentration in the culture medium, the root length gradually shortens, and on the 1 / 2MS + NAA 0.2mg / L + 0.5mg / L clofosinate medium, the root length is the shortest, which is 9.8cm.
[0024] 4) The results of the present invention show that the rooting frequency on the rooting medium without any growth regulator is the lowest, which is 73.3%, and the root system is sparse and weak. When 0.2 mg / L NAA is added, the rooting rate of the two rooting media reaches 100%, but the root system development of the 1 / 2MS basic medium is better than that of the MS basic medium, and the former has a more developed and longer root system. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a diagram of transplanting cherry sage tissue culture seedlings;
[0026] Figure 2 This is a picture of the new buds of cherry sage;
[0027] Figure 3This is a picture of the first generation of sterile tissue culture seedlings of cherry sage;
[0028] Figure 4 This is a picture of the result of bud multiplication and new bud induction;
[0029] Figure 5 This is a graph showing the effect of the concentration of clofosconazole in the culture medium on the root length of cherry sage seedlings (A is the result of bottom rooting with different hormones; B is the result of side rooting with different hormones). DETAILED DESCRIPTION
[0030] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.
[0031] The cherry sage material used in this application was planted in the herb resource garden of Shanghai Chenshan Botanical Garden.
[0032] Preparation of sterile water: Clean a 500mL reagent bottle, fill it with no more than 400mL of distilled water, cover it with a lid, and place it in a high-pressure steam sterilizer to sterilize it together with the culture medium to obtain sterile water.
[0033] Preparation of sterile culture dishes: Clean a 12cm diameter culture dish, cut the tracing paper used for engineering drawings into several circular pieces of paper slightly smaller than 12cm in diameter with scissors and place them in the dish, wrap them with newspapers and put them in a high pressure steam sterilizer to sterilize them together with the culture medium, and you can get a sterile culture dish with sterile paper. When inoculating and cutting tissue culture plant materials, change the sterile circular pieces of paper frequently to minimize bacterial and fungal contamination.
[0034] The preparation method of the culture medium in the following examples is as follows: weigh sucrose into a beaker, add a small amount of distilled water, MS basal culture medium powder and growth regulator mother solution. After stirring in the beaker, add an appropriate amount of distilled water, and use a pipette to drip a few drops of 1M NaOH and 1M HCL to adjust the pH value to 5.8-6. Usually 5 drops of 1M NaOH are needed. If the adjustment is too much, add HCL to adjust it back. After the pH value is adjusted, pour it into a special iron pot for an induction cooker, add agar powder when it is almost boiled on the induction cooker, stir with a glass rod until the liquid is transparent and the agar powder particles are invisible, pour it back into the beaker, and add distilled water to the original volume to make up for the distilled water lost when boiling the culture medium. The above culture media are all added with 30g / L sucrose and 7g / L agar.
[0035] Example 1
[0036] 1. Explant disinfection
[0037] The cherry sage plants from the Herb Resource Garden of Shanghai Chenshan Botanical Garden were transplanted into pots and placed in the laboratory for one month. After new branches sprouted from the old branches, the new branches were cut off for tissue culture ( Figure 1 ). New shoots grown in the laboratory are less likely to have endophytes inside, making them easier to sterilize.
[0038] Remove the cherry sage shoots and disinfect them with 70% ethanol solution for 0.5 minutes, then soak them in 0.1% mercuric chloride for 5 minutes, and rinse them with sterile water for 3-4 times. After rinsing, move the shoots to the clean bench and place them in a culture dish covered with sterile paper for cutting. Cut the shoots into stem segments with 1-2 leaves, lay them flat on the corresponding solid culture medium, and transport them to a culture room with a temperature of 25-28°C, a light intensity of 1500-3000Lux, and a 12h light and 12h dark photoperiod for cultivation. In order to prevent individual explants in one culture dish from being infected with bacteria by other explants due to incomplete sterilization, only two stem segments are inoculated in each culture dish.
[0039] 2. Primary Culture
[0040] The potted cherry sage was placed indoors for more than one month, and the newly grown shoots were cut, sterilized and inoculated into the new shoot induction medium (MS+NAA 0.1mg / L+BA 2mg / L). After two weeks, new shoots of cherry sage were clearly seen growing from the leaf axils of the stem segments ( Figure 2 After the new shoots are induced, they are transferred to the rooting medium (MS+NAA0.1mg / L+BA 0.2mg / L) for subculture to obtain the primary sterile seedlings ( Figure 3 ).
[0041] Example 2
[0042] 1. Effect of cytokinins on the rooting of cherry sage
[0043] During the primary culture process, cherry sage is very easy to root. Even if the culture medium contains cytokinin BA, it cannot prevent the tissue culture seedlings from growing new roots. Considering that the growth regulator NAA is also added to the culture medium, the role of the auxin in the culture medium cannot be completely ruled out. Therefore, a new shoot induction culture medium with different concentrations of cytokinin BA was designed for screening experiments.
[0044] The young shoots of the cherry sage tissue culture seedlings were cut into stem segments with 1-2 leaves, inoculated in the new shoot induction medium shown in Table 1 for culture, and 3 to 5 inoculated explants were placed in each culture bottle in triplicate. The germination rate = the number of explants that sprouted / the number of explants that were inoculated × 100%.
[0045] The results are shown in Table 1. The cherry sage seedlings took root 100% in the new shoot induction medium without cytokinin BA; new roots still grew in the new shoot induction medium with a lower concentration of cytokinin BA; with the continuous increase of cytokinin concentration, the rooting rate became lower and lower until the BA concentration reached 2 mg / L, the rooting rate was zero, indicating that BA in the new shoot induction medium has an inhibitory effect on rooting.
[0046] Table 1 Comparison of different cytokinin concentrations on the rooting rate of cherry sage stem segments
[0047] Culture medium Number of vaccinations Number of roots Rooting rate% MS+NAA0.1mg / L 30 30 100 MS+NAA0.1mg / L+BA0.1mg / L 30 29 96.7 MS+NAA0.1mg / L+BA0.2mg / L 30 25 83.3 MS+NAA0.1mg / L+BA0.5mg / L 30 16 53.3 MS+NAA0.1mg / L+BA1mg / L 30 8 26.7 MS+NAA0.1mg / L+BA2mg / L 30 0 0
[0048] 2. Effect of cytokinins on the induction of new shoots of Sage serrata
[0049] The young branches of the tissue culture seedlings of Sage cerasifera were cut into stem segments with 1-2 leaves, inoculated in the new shoot induction medium shown in Table 2, and cultured for 30 days, and then the new shoot induction rate was calculated.
[0050] The results are shown in Table 2. As the BA concentration increases, the bud induction rate gradually increases. When the BA concentration is 2 mg / L, the bud induction rate is the highest, which is 100%. New buds are produced in the leaf axils. When the BA concentration is low, there are only new single buds. When the BA concentration is greater than 1 mg / L, clustered buds can be seen growing in the leaf axils of many stem segments ( Figure 4 ).
[0051] Table 2 Comparison of different cytokinin concentrations on the bud induction rate of cherry sage stem segments
[0052]
[0053]
[0054] 3. Effect of uniconazole concentration on seedling height and root length of cherry sage
[0055] Another characteristic of cherry sage tissue culture seedlings is that they grow tall very easily. In some culture medium treatments, the height of the seedlings can reach the bottle mouth in 2-3 weeks. Therefore, a rooting medium with the plant growth retardant uniconazole was designed for screening tests. The mechanism of action of uniconazole is to inhibit the concentration of endogenous gibberellins in the plant body, thereby inhibiting the plant height and root length.
[0056] After clustered buds grew in the leaf axils of the stem segments, they were transferred to the rooting medium shown in Table 2 for subculture.
[0057] The results are shown in Table 3 and Figure 5As shown in the figure, with the increase of the concentration of clofosconazole in the culture medium, the seedling height gradually decreased. On the 1 / 2MS+NAA0.2mg / L+clofosconazole 0.5mg / L culture medium, the seedling height was the shortest, which was 7.7cm. With the increase of the concentration of clofosconazole in the culture medium, the root length gradually shortened. On the 1 / 2MS+NAA0.2mg / L+clofosconazole 0.5mg / L culture medium, the root length was the shortest, which was 9.8cm. In summary, the reason why cherry sage tissue culture seedlings tend to grow wildly is that the concentration of endogenous gibberellins in the plant is high.
[0058] Table 3 Effects of different uniconazole concentrations on the root length of cherry sage seedlings
[0059] Medium composition Average seedling height cm Average root length cm 1 / 2MS+NAA0.2mg / L 12.7 15.3 1 / 2MS+NAA0.2mg / L+vinthiophene 0.05mg / L 11.3 13.1 1 / 2MS+NAA0.2mg / L+vinthiophene 0.1mg / L 10.1 12.7 1 / 2MS+NAA0.2mg / L+vinthiophene 0.2mg / L 8.6 10.5 1 / 2MS+NAA0.2mg / L+vinthiophene 0.5mg / L 7.7 9.8
[0060] 4. Effects of different culture medium components on the rooting of cherry sage
[0061] After clustered buds grew in the leaf axils of the stem segments, they were transferred to the rooting medium shown in Table 4 for subculture.
[0062] The results are shown in Table 4. The rooting frequency on the rooting medium without any growth regulator was the lowest, at 73.3%, and the root system was sparse and weak. With the addition of 0.2 mg / L NAA, the rooting rate of both rooting media reached 100%, but the root system development of 1 / 2MS as the basic medium was better than that of MS as the basic medium, and the former had a more developed and longer root system.
[0063] Table 4 Effects of different rooting medium formulas on the rooting of cherry sage
[0064]
[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for tissue culture and rapid propagation of cherry sage, characterized in that: The method comprises the following steps: sterilizing and disinfecting the tender branches of cherry sage and then inoculating them into a new bud induction medium to induce new buds; and then transferring them to a rooting medium for subculture to obtain sterile seedlings of cherry sage.
2. The tissue culture rapid propagation method according to claim 1, characterized in that: The formula of the new sprout induction medium is: MS+0.1 mg / L NAA+0.1-2 mg / L BA+30 g / L sucrose+7 g / L agar.
3. The tissue culture rapid propagation method according to claim 2, characterized in that: The formula of the new sprout induction medium is: MS+0.1 mg / L NAA+2 mg / L BA+30 g / L sucrose+7 g / L agar.
4. The tissue culture rapid propagation method according to claim 1, characterized in that: The formula of the rooting medium is: 1 / 2MS or MS+0.2mg / L NAA+0.05-0.5mg / L uniconazole+30g / L sucrose+7g / L agar.
5. The tissue culture and rapid propagation method according to claim 4, characterized in that: The formula of the rooting medium is: 1 / 2MS or MS+0.2mg / L NAA+0.2mg / L uniconazole+30g / L sucrose+7g / L agar.
6. The tissue culture rapid propagation method according to claim 5, characterized in that: The formula of the rooting medium is: 1 / 2MS+0.2mg / L NAA+0.2mg / L uniconazole+30g / L sucrose+7g / L agar.
7. The tissue culture and rapid propagation method according to claim 5, characterized in that: The sterilization method comprises the following steps: sterilizing the cherry sage twigs with a 70% ethanol solution for 0.5 minutes, then soaking them in 0.1% mercuric chloride for 5 minutes, and then washing them with sterile water for 3-4 times.
8. The tissue culture and rapid propagation method according to any one of claims 1 to 6, characterized in that: The specific steps include: 1) Transplant the cherry sage plants grown outdoors into pots and place them in the laboratory for one month. When new shoots emerge from the old shoots, cut off the new shoots for tissue culture; 2) Sterilize the cherry sage shoots with 70% ethanol solution for 0.5 min, then soak them in 0.1% mercuric chloride for 5 min, and rinse them with sterile water 3-4 times after soaking; 3) After rinsing, the shoots are moved to a clean workbench and placed on a culture dish covered with sterile paper for cutting. The shoots are cut into stem segments with 1-2 leaves, inoculated into a new shoot induction medium, and placed in a culture room with a temperature of 25-28° C., a light intensity of 1500-3000 Lux, and a 12-hour light and 12-hour dark photoperiod for new shoot induction culture; 4) After new shoots grow out of the stem segments, they are inoculated into a rooting medium and placed in a culture room with a temperature of 25-28° C., a light intensity of 1500-3000 Lux, and a photoperiod of 12 h light and 12 h dark for rooting culture, thereby finally obtaining complete sterile cherry sage seedlings.