A tissue culture rapid propagation method of queen turmeric

Abstract

The application discloses a tissue culture and rapid propagation method of queen curcuma. By adding 4-8 mg / L ethoxyquin in the induction medium, the proliferation medium and the rooting and seedling strengthening medium, the germination of the dormant buds of the rhizome of the queen curcuma is promoted, the germination, proliferation and rooting and seedling strengthening of the bud body are promoted, and the germination rate, the proliferation multiple and the growth of the root system and the leaf of the bud are obviously improved. The application effectively expands the explant source of the queen curcuma and provides reliable and efficient technical guidance for the large-scale production of the queen curcuma tissue culture seedlings.

Description

Technical Field

[0001] This invention belongs to the field of plant cultivation technology, specifically relating to a method for rapid tissue culture propagation of Queen Tulip. Background Technology

[0002] Queen Tulip ( Curcuma petiolata *Curcuma longa* is a perennial herb belonging to the genus *Curcuma* in the family Zingiberaceae. It is native to Thailand and Malaysia, and has been introduced and cultivated in Guangdong Province. The plant grows to a height of 0.7-1.2 meters, with underground rhizomes that are ovoid to ovate, about 3.5 cm in diameter. The leaves are green, oblong-elliptic, 20-70 cm long and 15-35 cm wide, with long, soft hairs on the underside and pinnately arranged parallel veins. The cylindrical spike-like inflorescence emerges from between the leaf sheaths, 35-45 cm long and about 8 cm in diameter. The bracts are ovoid, 4-6 cm long, with the lower and middle bracts white to yellowish-green and the apex pale purplish-red; the calyx is white with three lobes at the apex; the corolla is golden yellow. It flowers from late spring to autumn.

[0003] Queen Tulip has a beautiful shape and elegant, uniquely colored bracts, making it suitable for planting in gardens or as a large potted plant. A single inflorescence can be enjoyed for more than a month. Its cut flowers are high-end floral material, and can be displayed in a vase for up to half a month. When it is not flowering, it can also be used as a foliage plant for indoor decoration.

[0004] Queen Tulip is typically propagated by dividing its rhizomes, but this method results in low propagation rates, slow growth, and a tendency to cause germplasm degradation, hindering large-scale production and restricting its industrial development. In contrast, tissue culture-based rapid propagation technology offers advantages such as a short production cycle, low material consumption, and stable traits, effectively compensating for the shortcomings of traditional propagation methods. This technology can solve the problems of slow propagation speed and germplasm degradation in Queen Tulip, providing a large number of uniform, high-quality seedlings for large-scale production, and has significant value for industrial applications.

[0005] The establishment of a rapid tissue culture propagation system begins with the selection of explants. Explants are small sections of tissue or organs excised from a plant for tissue culture. Different sampling sites and their physiological states are key factors affecting the success of tissue culture. For Curcuma species, buds from rhizomes are commonly used as explants. Rhizomes are short, segmented stem structures bearing numerous buds. Based on their growth state, these buds can be divided into two categories: active buds, including shoot tips and sprouting buds; and dormant buds, which are suppressed by apical dormancy. In Curcuma tissue culture, vigorous shoot tips and sprouting buds are usually preferred as explants, as these materials adapt to the tissue culture environment quickly after inoculation and resume growth; dormant buds, on the other hand, often remain dormant after inoculation and are difficult to initiate growth. Therefore, using active buds as explants increases the likelihood of successful tissue culture. However, the number of shoot tips and sprouts is significantly limited: a rhizome typically has only one shoot tip and one or two sprouts, resulting in limited material availability and hindering the large-scale development of rapid tissue culture propagation. To fully utilize the buds on rhizomes and overcome the limitation on the number of explants, utilizing dormant buds has become an effective solution. This invention uses dormant buds of *Tulipa chinensis* as explants. By adding specific agents to the culture medium, dormant buds are encouraged to adapt to the tissue culture environment and regain their growth vitality, thereby improving the utilization rate of explants and establishing a highly efficient rapid tissue culture propagation system. To date, there are no reports or related patents regarding the use of dormant buds of *Tulipa chinensis* for tissue culture.

[0006] Ethoxyquinoline (EQ) is a compound with antioxidant activity, commonly used in the production of oils, fishmeal, animal by-products, and animal feed. It can scavenge free radicals generated during feed and food storage, thus maintaining the quality of feed and its raw materials and ensuring feed safety. Currently, there are no reports or patent applications regarding the application of ethoxyquinoline in plant cultivation technology. Summary of the Invention

[0007] To address the shortcomings and deficiencies of existing technologies, this invention aims to provide a highly efficient method for rapid tissue culture propagation of Queen Tulips. This method effectively relieves the inhibition of dormant bud germination, significantly increases the germination rate and proliferation multiple, and promotes leaf and root growth, thereby establishing a stable and efficient tissue culture propagation system and providing reliable technical support for the large-scale production of Queen Tulip seedlings.

[0008] The first objective of this invention is to provide the application of ethoxyquinoline in improving the efficiency of Queen Turmeric tissue culture.

[0009] Preferably, the application is the application of ethoxyquinoline in improving the germination efficiency of dormant buds of Queen Tulip, promoting the proliferation of Queen Tulip buds, promoting the leaf growth of Queen Tulip seedlings, or promoting the root growth of Queen Tulip seedlings.

[0010] A second objective of this invention is to provide a culture medium that improves the efficiency of tissue culture of Queen Tulip, containing 2-10 mg / L of ethoxyquinoline.

[0011] Preferably, the culture medium is an induction medium, a proliferation medium, or a rooting and seedling strengthening medium.

[0012] Preferably, the induction medium contains 6-8 mg / L of ethoxyquinoline, the proliferation medium contains 6-8 mg / L of ethoxyquinoline, and the rooting and seedling strengthening medium contains 4-8 mg / L of ethoxyquinoline.

[0013] Preferably, the induction medium has the following composition: MS + TDZ 0.1-0.2 mg / L + EQ 6-8 mg / L + sucrose 25-30 g / L + agar 6-7 g / L, pH 5.8-6.0; The proliferation medium is composed of: MS + TDZ 0.3-0.5 mg / L + EQ 6-8 mg / L + sucrose 25-30 g / L + 6-7 g / L agar, pH 5.8-6.0; The composition of the rooting and seedling strengthening culture medium is: MS + NAA 0.1-0.5 mg / L + EQ 4-8 mg / L + sucrose 25-30 g / L + agar 6-7 g / L, pH 5.8-6.0.

[0014] The third objective of this invention is to provide a rapid tissue culture propagation method for Queen Tulip, comprising the following steps: S1. Cut dormant buds from the rhizomes of Queen Tulip, clean and disinfect them, and inoculate them into an induction medium to obtain germinating buds; the composition of the induction medium is: MS + TDZ 0.1-0.2 mg / L + EQ 6-8 mg / L + sucrose 25-30 g / L + agar 6-7 g / L, pH 5.8-6.0; S2. Inoculate the germinating shoots into a shoot proliferation medium to induce the formation and proliferation of clustered shoots. The composition of the proliferation medium is: MS + TDZ 0.3-0.5 mg / L + EQ 4-8 mg / L + sucrose 25-30 g / L + agar 6-7 g / L, pH 5.8-6.0; S3. The clustered buds are divided into individual plants and cultured in a rooting and seedling strengthening medium until rooted seedlings are obtained. The rooting medium consists of: MS + NAA 0.1-0.5 mg / L + EQ 4-8 mg / L + sucrose 25-30 g / L + agar 6-7 g / L, pH 5.8-6.0.

[0015] Preferably, the cleaning and disinfection steps in step S1 are as follows: rinse the dormant buds with water for 3-5 minutes, soak the buds in a 75% ethanol aqueous solution in a sterile environment for 0.5-1 minutes, and soak them in sterile water for 5 minutes; soak them in a 5% sodium hypochlorite aqueous solution for 10-15 minutes; and soak them in sterile water 3-5 times, each time for 5-8 minutes.

[0016] Preferably, the culture is carried out under the conditions of a culture temperature of 25±2℃, a light exposure time of 12-16 h / d, and a light intensity of 1500-2000 Lux.

[0017] The beneficial effects of this invention are: 1. Advantages of explant source: This invention uses dormant buds as explants. Compared with the active buds commonly used in Curcuma genus, there are more dormant buds, resulting in a richer source of materials. This effectively breaks through the limitations of material sources in tissue culture and is conducive to promoting large-scale tissue culture seedlings.

[0018] 2. Adding ethoxyquinoline to the induction medium significantly promotes the germination of dormant buds. In a comparative experiment, the germination rate of buds with 8 mg / L ethoxyquinoline reached 69.4%; in contrast, the germination rate of buds in the medium without ethoxyquinoline was less than 20%.

[0019] 3. Adding ethoxyquinoline to the proliferation medium can effectively promote the formation and proliferation of shoot clusters, significantly increasing the bud proliferation rate. In a comparative experiment, adding 8 mg / L ethoxyquinoline increased the proliferation rate to 8.5 times, while without EQ, the proliferation rate was only 4.2 times.

[0020] 4. Adding ethoxyquin to the rooting and seedling strengthening medium can effectively promote the growth of leaves and roots. In a comparative experiment, when 8 mg / L of ethoxyquin was added, the average height of the tissue culture seedlings reached 9.63 cm, the average number of roots was 4.93, the average root diameter was 1.76 mm, and the average root length was 3.44 cm; while without the addition of ethoxyquin, the average height of the seedlings was 6.48 cm, the average number of roots was 3.13, the average root diameter was 1.11 mm, and the average root length was 1.24 cm.

[0021] In summary, this invention overcomes the technical difficulties of existing technologies in using dormant buds, which are difficult to germinate and proliferate, thus preventing efficient propagation, by adding ethoxyquinoline (EQ) to the culture medium required for each stage of tissue culture. It systematically improves the efficiency and quality of each stage of Queen Tulip tissue culture and provides reliable technical support for achieving efficient and stable large-scale seedling production. Attached Figure Description

[0022] Figure 1 These are different types of buds on the rhizome.

[0023] Figure 2 This refers to the effect of ethoxyquinoline on the germination of dormant buds.

[0024] Figure 3 The results show the effects of different concentrations of ethoxyquinoline on the proliferation of shoot clusters; where EQ0, EQ2, EQ4, EQ6, EQ8, and EQ10 represent ethoxyquinoline concentrations of 0, 2, 4, 6, 8, and 10 mg / L, respectively.

[0025] Figure 4 The results show the effects of different concentrations of ethoxyquinoline on the leaf and root growth of tissue culture seedlings of Queen Tulip; where EQ0, EQ2, EQ4, EQ6, EQ8, and EQ10 represent ethoxyquinoline concentrations of 0, 2, 4, 6, 8, and 10 mg / L, respectively. Detailed Implementation

[0026] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0027] The following examples are tissue culture experiments conducted using Queen Tulip as the experimental subject, and the purity of the ethoxyquinoline (EQ) used was 99%.

[0028] Example 1 1. Bud Disinfection and Induction: Dormant buds from rhizomes were used as explants. After rinsing with running water for 5 minutes, the buds were soaked in a 75% ethanol aqueous solution for 1 minute, followed by a 5-minute soak in sterile water on a clean bench. Then, the buds were soaked in a 5% sodium hypochlorite aqueous solution for 15 minutes, with continuous shaking to ensure thorough disinfection. Finally, the explants were soaked in sterile water five times, 5 minutes each time, to remove any residual sodium hypochlorite. The disinfected buds were inoculated into an induction medium and germination was induced at a temperature of 25±2℃, a light duration of 16 h / d, and a light intensity of 2000 Lux. After 30 days, the germination rate was 68.8%, and the germinating buds were considered germinating buds. The induction medium consisted of: MS + TDZ 0.2 mg / L + EQ 8 mg / L + sucrose 30 g / L + agar 7 g / L, pH 5.8.

[0029] 2. Induction and proliferation culture of clustered shoots: The germinated shoots obtained above were transferred to a proliferation medium to induce the formation and proliferation of clustered shoots. The shoots were cultured for 30 days at a temperature of 25±2℃, a light duration of 16 h / d, and a light intensity of 2000 Lux. The fold increase of the shoots was statistically analyzed to be 8.4. The proliferation medium consisted of: MS + TDZ 0.5 mg / L + EQ 8 mg / L + sucrose 30 g / L + agar 7 g / L, pH 5.8.

[0030] 3. Rooting and Seedling Strengthening Culture: The clustered buds obtained above were cut into single plants and transferred to a rooting and seedling strengthening culture medium. The plants were cultured for 30 days at a temperature of 25±2℃, a light duration of 16 h / d, and a light intensity of 2000 Lux to obtain rooted seedlings. The rooting rate was 100%, with an average seedling height of 9.56 cm, an average number of roots of 4.90, a root diameter of 1.82 mm, and a root length of 3.42 cm. The seedlings were robust. The rooting and seedling strengthening culture medium consisted of: MS + NAA 0.5 mg / L + EQ 8 mg / L + sucrose 30 g / L + agar 7 g / L, pH 5.8.

[0031] Example 2 1. Bud Disinfection and Induction: Dormant buds from rhizomes were used as explants. After rinsing with running water for 4 minutes, the buds were soaked in a 75% ethanol aqueous solution for 0.5 minutes and then in sterile water for 5 minutes on a clean bench. Next, the buds were soaked in a 5% sodium hypochlorite aqueous solution for 12 minutes, with constant shaking to ensure thorough disinfection. Afterward, the explants were soaked in sterile water four times, 8 minutes each time, to remove any residual sodium hypochlorite. The disinfected buds were inoculated into an induction medium and germination was induced at a temperature of 25±2℃, a light duration of 14 h / d, and a light intensity of 1500 Lux. After 30 days, the germination rate was 66.7%, and the germinating buds were considered germinating buds. The induction medium consisted of: MS + TDZ 0.1 mg / L + EQ 7 mg / L + sucrose 30 g / L + agar 6.5 g / L, pH 5.9.

[0032] 2. Induction and proliferation culture of clustered shoots: The germinated shoots obtained above were transferred to a proliferation medium to induce the formation and proliferation of clustered shoots. The shoots were cultured for 30 days at a temperature of 25±2℃, a light duration of 14 h / d, and a light intensity of 1500 Lux. The fold increase of the shoots was statistically analyzed to be 8.2. The proliferation medium consisted of: MS + TDZ 0.4 mg / L + EQ 7 mg / L + sucrose 30 g / L + agar 6.5 g / L, pH 5.9.

[0033] 3. Rooting and Seedling Strengthening Culture: The clustered buds obtained above were cut into single plants and transferred to a rooting and seedling strengthening culture medium. The plants were cultured for 30 days at a temperature of 25±2℃, a light duration of 14 h / d, and a light intensity of 1500 Lux. The rooting rate was 100%, with an average seedling height of 9.44 cm, an average number of roots of 4.83, a root diameter of 1.75 mm, and a root length of 3.62 cm. The seedlings were robust. The rooting and seedling strengthening culture medium consisted of: MS + NAA 0.3 mg / L + EQ 7 mg / L + sucrose 30 g / L + agar 6.5 g / L, pH 5.9.

[0034] Example 3 1. Bud Disinfection and Induction: Dormant buds from rhizomes were used as explants. After rinsing with running water for 3 minutes, the buds were soaked in a 75% ethanol aqueous solution for 1 minute and then in sterile water for 5 minutes on a clean bench. Next, the buds were soaked in a 5% sodium hypochlorite aqueous solution for 10 minutes, with constant shaking to ensure thorough disinfection. Afterward, the explants were soaked in sterile water three times, 5 minutes each time, to remove any residual sodium hypochlorite. The disinfected buds were inoculated into an induction medium and germination was induced at a temperature of 25±2℃, a light duration of 12 h / d, and a light intensity of 2000 Lux. After 30 days, the germination rate was 62.9%, and the germinating buds were considered germinating buds. The induction medium consisted of: MS + TDZ 0.1 mg / L + EQ 6 mg / L + sucrose 25 g / L + agar 6 g / L, pH 6.0.

[0035] 2. Induction and proliferation culture of clustered shoots: The germinated shoots obtained above were transferred to a proliferation medium to induce the formation and proliferation of clustered shoots. The shoots were cultured for 30 days at a temperature of 25±2℃, a light duration of 12 h / d, and a light intensity of 2000 Lux. The fold increase of the shoots was statistically analyzed to be 8.0. The proliferation medium consisted of: MS + TDZ 0.3 mg / L + EQ 6 mg / L + sucrose 25 g / L + agar 6 g / L, pH 6.0.

[0036] 3. Rooting and Seedling Strengthening Culture: The clustered buds obtained above were cut into single plants and transferred to a rooting and seedling strengthening culture medium. The plants were cultured for 30 days at a temperature of 25±2℃, a light duration of 12 h / d, and a light intensity of 2000 Lux to obtain rooted seedlings. The rooting rate was 100%, with an average seedling height of 9.24 cm, an average number of roots of 4.75, a root diameter of 1.72 mm, and a root length of 3.51 cm. The seedlings were robust. The rooting and seedling strengthening culture medium consisted of: MS + NAA 0.1 mg / L + EQ 6 mg / L + sucrose 25 g / L + agar 6 g / L, pH 6.0.

[0037] Example 4 This embodiment examines the effects of different explant sources and EQ on tissue culture. The specific scheme is as follows: 1. Induction response of different explants Two types of buds exist on the rhizomes of Queen Tulip: one type is active buds, including shoot tips and sprouting buds, characterized by bud swelling and leaf emergence; the other type is dormant buds, which are flat and do not produce leaves. Figure 1 These two types of buds have different adaptability to the tissue culture environment. To compare their differences, this experiment selected shoot tips, sprouting buds, and dormant buds as explants for culture.

[0038] Following the steps described in Example 1, various buds were disinfected and then inoculated into an induction medium (composition as follows: MS + TDZ 0.2 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8) and cultured for 30 days. The contamination rate, mortality rate, survival rate, and germination rate of different explants were then statistically analyzed. The results are shown in Table 1. The contamination rates of shoot tips, germinating buds, and dormant buds were 33.3%, 31.1%, and 36.7%, respectively; the mortality rates were 11.2%, 8.9%, and 5.6%, respectively; and the survival rates were 55.5%, 60.0%, and 57.8%, respectively, with no significant differences. This indicates that different buds showed relatively consistent tolerance to disinfection conditions, and their survival rates after inoculation were similar. Regarding the germination performance of surviving buds, the three types of buds showed significant differences: the germination rates of shoot tips and sprouting buds were 62.1% and 64.7%, respectively, with more than half of the buds successfully germinating, producing leaves, and elongating. In contrast, the germination rate of dormant buds was only 17.5%, while the remaining 82.5% of dormant buds remained stagnant, without producing leaves. This result indicates that conventional culture medium formulations are difficult to effectively induce the germination and growth of dormant buds, leading to the underutilization of this bud resource.

[0039] Table 1. Induction responses of different explants Note: All results were statistically analyzed 30 days after inoculation. The values ​​are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (P<0.05), and the same lowercase letters after the data in the same column indicate no significant differences (P>0.05). Contamination rate = (number of contaminated buds / total number of inoculated buds) × 100%; Mortality rate = (number of dead buds / total number of inoculated buds) × 100%; Survival rate = (number of surviving buds / total number of inoculated buds) × 100%; Germination rate = (number of germinated buds / total number of surviving buds) × 100%.

[0040] 2. Induction of dormant shoots in Queen Tulip by different concentrations of ethoxyquinoline To address the problem of limited explants caused by the difficulty in germinating dormant buds after inoculation, this experiment used dormant buds as explants and added different concentrations of ethoxyquinoline to the culture medium to explore its effect on the germination of dormant buds, providing a basis for establishing an efficient tissue culture rapid propagation system.

[0041] Dormant buds were sterilized according to the method in Example 1 and inoculated into induction medium supplemented with different concentrations of ethoxyquinoline (specific composition: MS + TDZ 0.2 mg / L + EQ 0-10 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8) to investigate the promoting effect of ethoxyquinoline on the germination of dormant buds. The results are shown in Table 2. In the control medium without ethoxyquinoline, only 18.1% of the dormant buds were able to germinate, while the remaining 81.9% of the buds remained in a dormant state at the time of inoculation and could not resume growth; however, the addition of ethoxyquinoline promoted the germination of dormant buds, resulting in leaf growth and bud elongation. Figure 2 At a concentration of 2 mg / L ethoxyquinoline, 35.3% of dormant buds successfully germinated; at 4 mg / L, 53.2% of dormant buds successfully germinated; when the concentration of ethoxyquinoline increased to 6 mg / L, the germination rate of dormant buds reached 63.6%; further increasing the concentration of ethoxyquinoline to 8 mg / L resulted in a germination rate of 69.4%, which was not significantly different from the 6 mg / L treatment; when the concentration of ethoxyquinoline reached 10 mg / L, the germination rate of dormant buds decreased to 55.1%, indicating that excessively high concentrations of ethoxyquinoline could not further increase the germination rate, and that 6-8 mg / L was the suitable concentration range for breaking the dormancy of Queen Tulip buds.

[0042] Table 2 Effects of ethoxyquinoline on the germination of dormant shoots of Queen Tulip Note: All results were statistically analyzed 30 days after inoculation. The values ​​are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (P<0.05), and the same lowercase letters after the data in the same column indicate no significant differences (P>0.05); Germination rate = (number of germinated buds / number of surviving buds) × 100%.

[0043] 3. Effects of different ethoxyquinolines on the proliferation of adventitious shoots of Queen Tulip Germinating shoots induced on induction medium without ethoxyquinoline (composition: MS + TDZ 0.2 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8) were transferred to proliferation media with different concentrations of ethoxyquinoline (composition: MS + TDZ 0.5 mg / L + EQ 0-10 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8) to investigate the effect of ethoxyquinoline on shoot proliferation. Culture was performed under the same conditions as in Example 1, and the results are shown in Table 3. In the control medium without ethoxyquinoline, the fold increase of shoots was only 4.2. After adding ethoxyquinoline to 2 mg / L, the fold increase significantly increased to 6.4, indicating that ethoxyquinoline has a significant promoting effect on bud proliferation. The fold increase continued with increasing ethoxyquinoline concentration, reaching a maximum of 8.5 at a concentration of 8 mg / L, but the difference between this and the 6 mg / L treatment was not statistically significant. When the ethoxyquinoline concentration increased to 10 mg / L, the fold increase decreased. Figure 3 The results showed that, within the experimental concentration range, 6-8 mg / L of ethoxyquinoline was the optimal concentration range for inducing the proliferation of adventitious shoots in Queen Tulip.

[0044] Table 3. Effects of ethoxyquinoline on the proliferation of Queen Tulip buds Note: All results were statistically analyzed 30 days after inoculation. The values ​​are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (P<0.05), and the same lowercase letters after the data in the same column indicate no significant differences (P>0.05); Proliferation ratio = number of proliferating buds / number of inoculated buds.

[0045] 4. Effects of different ethoxyquinolines on rooting and seedling vigor of Queen Tulip Clustered shoots obtained from cultivation in a growth medium without ethoxyquin (composition: MS + TDZ 0.5 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8) were separated into single shoots and transferred to rooting and seedling strengthening media with different concentrations of ethoxyquin (MS + NAA 0.5 mg / L + EQ 0-10 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8) to allow for full leaf and root growth. Figure 4Following the culture conditions of Example 1, the effects of ethoxyquinoline on the leaf and root growth of tissue-cultured seedlings were investigated. The results are shown in Table 4: In the control medium without ethoxyquinoline, the average seedling height was 6.48 cm, the average number of roots was 3.13, the average root diameter was 1.11 mm, and the average root length was 1.24 cm. After the addition of ethoxyquinoline, all indicators increased significantly, indicating that ethoxyquinoline promoted the growth of leaves and roots. In terms of promoting seedling height, ethoxyquinoline at 6-8 mg / L achieved the most significant effect, with seedling height exceeding 9 cm. In terms of promoting root growth, ethoxyquinoline at 4-8 mg / L achieved the most significant effect, with an average number of roots of 4.73-5.07, an average root diameter of 1.76-1.84 mm, and an average root length of 3.44-3.76 cm. Therefore, in practical applications, if the goal is to promote leaf growth, it is recommended to use an ethoxyquin concentration of 6-8 mg / L; if the focus is on promoting root growth, an ethoxyquin concentration of 4-8 mg / L can be used.

[0046] Table 4. Effects of ethoxyquinoline on rooting of vigorous Queen Tulip seedlings Note: All results were statistically analyzed 30 days after vaccination. The values ​​are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (P<0.05), and the same lowercase letters after the data in the same column indicate no significant differences (P>0.05).

[0047] In summary, adding 4-8 mg / L of ethoxyquinoline to the induction medium, proliferation medium, and rooting and seedling strengthening medium can effectively break the dormancy of dormant buds of Queen Tulip, promote bud proliferation, and promote the growth of leaves and roots of tissue culture seedlings, providing reliable and efficient technical guidance for the large-scale production of Queen Tulip tissue culture seedlings.

Claims

1. The application of an induction culture medium in improving the germination efficiency of dormant buds in Queen Tulip, characterized in that, The induction medium consists of: MS + TDZ 0.1-0.2 mg / L + EQ 6-8 mg / L + sucrose 25-30 g / L + agar 6-7 g / L, pH 5.8-6.

0.

2. The application of a proliferation culture medium in promoting the proliferation of Queen Tulip buds, characterized in that, The proliferation medium is composed of: MS + TDZ 0.3-0.5 mg / L + EQ 6-8 mg / L + sucrose 25-30 g / L + agar 6-7 g / L, pH 5.8-6.

0.

3. The application of a rooting and seedling strengthening culture medium in promoting leaf growth or root growth of Queen Tulip seedlings, characterized in that, The composition of the rooting and seedling strengthening culture medium is: MS + NAA 0.1-0.5 mg / L + EQ 4-8 mg / L + sucrose 25-30 g / L + agar 6-7 g / L, pH 5.8-6.

0.

4. A rapid tissue culture propagation method for Queen Tulip, characterized in that, Includes the following steps: S1. Cut dormant buds from the rhizomes of Queen Tulip, clean and disinfect them, and inoculate them into an induction medium to obtain germinating buds; the composition of the induction medium is: MS + TDZ 0.1-0.2 mg / L + EQ 6-8 mg / L + sucrose 25-30 g / L + agar 6-7 g / L, pH 5.8-6.0; S2. Inoculate the germinating shoots into a shoot proliferation medium to induce the formation and proliferation of clustered shoots. The composition of the proliferation medium is: MS + TDZ 0.3-0.5 mg / L + EQ 4-8 mg / L + sucrose 25-30 g / L + agar 6-7 g / L, pH 5.8-6.0; S3. The clustered buds are divided into individual plants and cultured in a rooting and seedling strengthening medium until rooted seedlings are obtained. The rooting and seedling strengthening medium consists of: MS + NAA 0.1-0.5 mg / L + EQ 4-8 mg / L + sucrose 25-30 g / L + agar 6-7 g / L, pH 5.8-6.

0.

5. The method according to claim 4, characterized in that, The cleaning and disinfection steps in step S1 are as follows: rinse the dormant buds with water for 3-5 minutes, soak the buds in a 75% ethanol aqueous solution for 0.5-1 minute in a sterile environment, and soak them in sterile water for 5 minutes; soak them in a 5% sodium hypochlorite aqueous solution for 10-15 minutes; and soak them in sterile water 3-5 times, 5-8 minutes each time.

6. The method according to claim 4, characterized in that, The culture was carried out at a temperature of 25±2℃, a light exposure time of 12-16 h / d, and a light intensity of 1500-2000 Lux.

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