A method for promoting seed germination and adventitious bud regeneration of limonium aureum

Abstract

The application discloses a method for promoting seed germination and adventitious bud regeneration of Limonium aureum. The application uses a germination medium with MS medium+0.3mg / L 6-BA+0.9mg / L NNA+8.6mg / L ZnSO4 to carry out seed germination and seedling culture on Limonium aureum, and uses an adventitious bud induction medium with MS medium+0.3mg / L 6-BA+0.9mg / L NNA+0.32mg / L CuSO4 / 0.26mg / L ZnSO4 / 0.14mg / L AgNO3 to carry out adventitious bud induction. The application solves the problems of difficult seed breeding and difficult adventitious bud induction in the conventional culture method of Limonium aureum, and establishes an efficient, stable and rapid method for inducing differentiation of adventitious buds of explants for the tissue rapid propagation technology of Limonium aureum.

Description

Technical Field

[0001] This invention belongs to the field of plant cell engineering technology, specifically relating to a method for promoting the germination of *Limonium sinense* seeds and the regeneration of adventitious buds. Background Technology

[0002] Golden Limonium (Limonium aureum L.), also known as Golden Limonium, Golden Limonium, or Golden Spoonleaf Limonium, is a perennial herb belonging to the genus Limonium in the family Plumbaginaceae. It possesses excellent characteristics such as drought resistance, salt tolerance, and tolerance to poor soil. Its flowers are vibrant, abundant, and elegant, with a flowering period lasting from May to October. Even after the flowers dry in winter, the small golden-yellow blossoms can still be enjoyed for two months, making it a rare pure yellow perennial flower variety for landscaping. With its unique ecological adaptability, beautiful appearance, and medicinal value, it holds significant importance in botany and traditional Chinese medicine.

[0003] The yellow-flowered limonite is mainly distributed in parts of Northeast, North, and Northwest China, as well as Sichuan Province, including Heilongjiang, Jilin, Inner Mongolia, Hebei, Shanxi, and Sichuan. It typically grows in saline gravel beaches, loess slopes, sandy soils, plains, and the lower parts of hillsides, demonstrating its adaptation to saline soils and arid environments.

[0004] It also has important applications in the field of traditional Chinese medicine. Its calyx and root are commonly used folk herbs with analgesic, anti-inflammatory, and blood-tonifying effects. It is mainly used to treat symptoms such as neuralgia, scanty menstruation, tinnitus, insufficient lactation, and colds. Externally, it can also be used to treat toothache, boils, and carbuncles.

[0005] Currently, *Limonium sinense* is mostly found in the wild, and there are no standardized techniques for its artificial seedling cultivation and planting. The aseptic seedling propagation process for *Limonium sinense* is also quite lengthy, with a low germination rate; it takes at least 45 days from seed germination to a usable seedling. Simultaneously, the regeneration rate of adventitious buds from explants is low, and the regeneration cycle is long, severely hindering the aseptic seedling propagation and rapid tissue culture propagation of *Limonium sinense*. These problems can be solved using metal ion-promoted growth and germination techniques and tissue culture techniques. Through tissue culture propagation of *Limonium sinense*, efficient seedling propagation can be achieved, maintaining varietal characteristics, overcoming seasonal and geographical limitations, reducing production costs, and strengthening the preservation and utilization of germplasm resources. Therefore, researching standardized seedling cultivation and tissue culture techniques for *Limonium sinense* is of great significance for developing resilient native tree species, promoting national greening and healthy ecological development, and enhancing ecological, economic, and social benefits.

[0006] Traditional methods for inducing seed germination and adventitious bud formation in explants of Limonium stenoptera involve low seed germination rates, require specialized callus induction and subculture steps, and are prone to problems such as low adventitious bud induction rates, poor growth, and low seedling formation rates. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems of slow seed germination time of *Limonium sibiricum*, excessively long culture time, low regeneration rate, poor growth, and too many seedling induction steps (requiring subculture) in the process of inducing *Limonium sibiricum* explants to form adventitious buds. The invention provides a method for promoting the cultivation of *Limonium sibiricum* seedlings and inducing the differentiation of *Limonium sibiricum* petiole explants to obtain adventitious buds.

[0008] This invention utilizes zinc sulfate (ZnSO4) to promote seed germination of *Limonium sibiricum* and also provides a method for inducing adventitious bud formation in *Limonium sibiricum* explants: Copper sulfate (CuSO4) solution, zinc sulfate (ZnSO4) solution, and silver nitrate (AgNO3) solution are added to MS medium containing 0.3 mg / L 6-benzylaminopurine (6-BA) and 0.9 mg / L naphthaleneacetic acid (NAA). Then, the explants of *Limonium sibiricum* are inoculated into MS medium containing 0.3 mg / L 6-benzylaminopurine (6-BA) and 0.9 mg / L naphthaleneacetic acid (NAA) for adventitious bud induction. This invention can significantly improve the growth of aseptic seedlings of *Limonium sibiricum* and the efficiency of adventitious bud induction, accelerate the growth and differentiation of adventitious buds, and further shorten the culture cycle of *Limonium sibiricum*.

[0009] The first objective of this invention is to provide a germination culture medium for *Limonium sinense* seeds, comprising: 0.3 mg / L 6-BA, 0.9 mg / L NAA, 8.6 mg / L ZnSO4, with the remainder being MS medium.

[0010] Preferably, the MS culture medium has the following formulation: 16.5 g / L ammonium nitrate, 19 g / L potassium nitrate, 1.7 g / L potassium dihydrogen phosphate, 3.7 g / L magnesium sulfate heptahydrate, 4.4 g / L calcium chloride dihydrate, 16.9 mg / L manganese sulfate monohydrate, 8.6 mg / L zinc sulfate heptahydrate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L disodium molybdenum sulfate dihydrate, 0.025 mg / L sodium sulfate dihydrate, and 0.025 mg / L sodium sulfate dihydrate. The composition consists of: 0.025 mg / L copper sulfate pentahydrate, 0.025 mg / L cobalt chloride hexahydrate, 37.3 mg / L sodium ethylenediaminetetraacetate dihydrate, 27.8 mg / L ferrous sulfate heptahydrate, 2 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, 100 mg / L inositol, 30 g / L sucrose, 6 g / L agar, with the balance being water, and a pH of 5.8-6.0.

[0011] The second objective of this invention is to provide an adventitious bud induction culture medium for *Limonium sinense*, comprising any one of the following (1)-(3):

[0012] (1) 0.3 mg / L 6-BA, 0.9 mg / L NAA, 0.32 mg / L CuSO4, with the remainder being MS medium;

[0013] (2) 0.3 mg / L 6-BA, 0.9 mg / L NAA, 0.26 mg / L ZnSO4, with the remainder being MS medium;

[0014] (3) 0.3 mg / L 6-BA, 0.9 mg / L NAA, 0.14 mg / L AgNO3, with the remainder being MS medium.

[0015] Preferably, the MS culture medium has the following formulation: 16.5 g / L ammonium nitrate, 19 g / L potassium nitrate, 1.7 g / L potassium dihydrogen phosphate, 3.7 g / L magnesium sulfate heptahydrate, 4.4 g / L calcium chloride dihydrate, 16.9 mg / L manganese sulfate monohydrate, 8.6 mg / L zinc sulfate heptahydrate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L disodium molybdenum sulfate dihydrate, 0.025 mg / L sodium sulfate dihydrate, and 0.025 mg / L sodium sulfate dihydrate. The composition consists of: 0.025 mg / L copper sulfate pentahydrate, 0.025 mg / L cobalt chloride hexahydrate, 37.3 mg / L sodium ethylenediaminetetraacetate dihydrate, 27.8 mg / L ferrous sulfate heptahydrate, 2 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, 100 mg / L inositol, 30 g / L sucrose, 6 g / L agar, with the balance being water, and a pH of 5.8-6.0.

[0016] A third objective of this invention is to provide a method for inducing the differentiation of *Limonium sibiricum* into explants, comprising the following steps:

[0017] S1. After sterilization, the seeds of *Limonium sibiricum* were inoculated into the culture medium and germinated for 30 days to obtain sterile seedlings.

[0018] S2. Take the petioles of the sterile seedlings obtained in step S1 as explants and inoculate them into the adventitious bud induction medium to induce adventitious buds.

[0019] Preferably, the seeds mentioned in step S1 are conical, plump seeds without obvious lesions or mold.

[0020] Preferably, the disinfection process in step S1 involves soaking the seeds in a 75% ethanol aqueous solution for 60 seconds, rinsing them 2-3 times with sterile water, then soaking them in a 2% NaClO aqueous solution for 15 minutes with continuous shaking, and finally rinsing them 3-4 times with sterile water for 5 seconds each time.

[0021] Preferably, the petiole mentioned in step S2 is a petiole segment with a length of 1 cm after removing the bud point at the connection between the petiole and the stem segment.

[0022] The beneficial effects of this invention are:

[0023] This invention employs MS medium containing 6-BA and NAA with a low concentration of ZnSO4 to prepare a germination medium for seed germination and seedling culture. Explants from the seedlings are inoculated into MS medium containing low concentrations of CuSO4, ZnSO4, or AgNO3 solution and 6-BA and NAA hormones for adventitious bud induction from petiole explants. This effectively shortens the time for obtaining *Limonium sibiricum* seedlings and adventitious buds, improves the quantity, quality, and efficiency of adventitious bud production from *Limonium sibiricum* petiole explants, significantly shortens the time for obtaining sterile seedlings, and increases the regeneration rate and differentiation rate of adventitious buds. This invention solves the problems of difficult seed propagation of *Limonium sibiricum* and the difficulty in inducing adventitious buds using conventional culture methods. It establishes an efficient, stable, and rapid method for inducing adventitious bud differentiation from explants for rapid tissue propagation of *Limonium sibiricum*, further providing an effective method for the propagation of high-quality *Limonium sibiricum* seedlings. Attached Figure Description

[0024] Figure 1 The study investigated the effects of different concentrations of ZnSO4 added to the germination medium on the germination and growth of *Limonium sibiricum* seeds. The values ​​A, E, and E represent the germination status of *Limonium sibiricum* seeds 30 days after the addition of 0, 8.6, 17.2, 25.8, and 37.4 mg / L ZnSO4, respectively. The scale bars in A, E, and E are all 1.0 cm.

[0025] Figure 2 The study investigated the effect of different concentrations of CuSO4 added to the adventitious shoot induction medium on the induction of adventitious shoots from *Limonium sibiricum* petiole explants. AF represents the effect of adding 0, 0.16, 0.32, 0.64, 1.28, and 2.56 mg / L CuSO4 to *Limonium sibiricum* petiole explants for 18 days to induce adventitious shoot production. The scale bar in AE is 1.0 cm.

[0026] Figure 3 The study investigated the effects of additional ZnSO4 or AgNO3 supplementation on the induction of adventitious shoots from *Limonium sibiricum* petiole explants. In A, the effect was observed after 18 days of additional ZnSO4 supplementation on adventitious shoot induction from *Limonium sibiricum* petiole explants; in B, the effect was observed after 18 days of additional AgNO3 supplementation on adventitious shoot induction from *Limonium sibiricum* petiole explants. The scale bars in both A and B were 0.5 cm. Detailed Implementation

[0027] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0028] To accurately add low-concentration CuSO4, ZnSO4, AgNO3, and hormone solutions, stock solutions for each solution need to be prepared for dilution and addition. Accurately weigh 1000 mg of anhydrous copper sulfate, anhydrous zinc sulfate, and silver nitrate using an analytical balance. Dissolve them thoroughly in deionized water, then bring the volume to 1L to prepare 1000 mg / L CuSO4, ZnSO4, and AgNO3 solutions, respectively. Accurately weigh 100 mg of 6-benzylaminopurine (6-BA) and naphthaleneacetic acid (NAA) powder using an analytical balance. Dissolve them thoroughly in 1 mol / L NaOH solution, then bring the volume to 1L with deionized water. Filter and sterilize using a 0.22 μm filter membrane to prepare 100 mg / L 6-BA and 100 mg / L NAA solutions, respectively.

[0029] A certain amount of the 1000 mg / L CuSO4 solution, ZnSO4 solution, and AgNO3 solution prepared above were respectively added to the MS medium solution. The pH was then adjusted to 5.8 with 1 mg / L NaOH aqueous solution or 1 mg / L HCl aqueous solution. The solution was then sterilized at 121 °C and 0.1 MPa for 15 minutes. A certain amount of the 100 mg / L 6-BA solution and 100 mg / L NAA solution prepared above were added to the culture medium solution before sterilization and dispensing (i.e., the unsolidified culture medium) to make the final concentration of 6-BA in the culture medium 0.3 mg / L and the final concentration of NAA in the culture medium 0.9 mg / L. After thorough mixing, the culture medium was dispensed. Therefore, germination media were prepared by adding 0.3 mg / L 6-BA and 0.9 mg / L NAA to MS medium and additionally adding different concentrations of ZnSO4 (0, 8.6, 17.2, 25.8, 34.4 mg / L). Also, germination media were prepared by adding 0.3 mg / L 6-BA and 0.9 mg / L NAA to MS medium and additionally adding different concentrations of CuSO4 (0, 0.16, 0.32, 0.64, 1.28, 2.56 mg / L), adding 0.3 mg / L 6-BA and 0.9 mg / L NAA to MS medium and additionally adding different concentrations of ZnSO4 (0, 0.13, 0.26, 0.52, 1.04, 2.08 mg / L), or adding 0.3 mg / L 6-BA and 0.9 mg / L NAA to MS medium. Adventitious shoot induction media containing NAA and additionally supplemented with different concentrations of AgNO3 (0, 0.14, 0.42, 1.00, 1.57, 3.15 mg / L). All media must be solidified and cooled to room temperature before use. The MS medium formulation is as follows: Inorganic components (16.5 g / L ammonium nitrate, 19 g / L potassium nitrate, 1.7 g / L potassium dihydrogen phosphate, 3.7 g / L magnesium sulfate heptahydrate, 4.4 g / L calcium chloride dihydrate, 16.9 mg / L manganese sulfate monohydrate, 8.6 mg / L zinc sulfate heptahydrate, 6.2 mg / L boric acid, 0.83 mg / L potassium iodide, 0.25 mg / L disodium molybdenum sulfate dihydrate, 0.0... The mixture consists of 25 mg / L copper sulfate pentahydrate, 0.025 mg / L cobalt chloride hexahydrate, 37.3 mg / L sodium EDTA dihydrate, and 27.8 mg / L ferrous sulfate heptahydrate, plus organic components (2 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, and 0.5 mg / L nicotinic acid), 100 mg / L inositol, 30 g / L sucrose, and 6 g / L agar, with the remainder being water.

[0030] The petiole explants used in Examples 2-4 were all taken from sterile seedlings grown for 30 days under the optimal germination conditions in Example 1. The connection between the petiole and the stem segment was severed, the bud at the connection point was removed, the leaf blade was cut off, and the petiole was retained. The petiole was cut into 1cm segments as petiole explants. The petiole wounds were exposed to sterile air. The petiole explants needed to be inoculated into the adventitious bud induction medium within 5-10 minutes to prevent the explants from losing water and becoming inactive due to prolonged exposure to hot sterile air.

[0031] All data were analyzed using SPSS Statistics 26.0 software for ANOVA and Duncan's multiple comparisons. Different letters after the data indicate significant differences between treatments (P≦0.05).

[0032] Example 1: Effects of germination media containing different concentrations of ZnSO4 on seed germination and seedling growth of Limonium sibiricum

[0033] Take a small amount of *Limonium sinense* seeds, remove the seed coat and other impurities, and select cone-shaped, plump seeds without obvious lesions or mold. Soak the seeds in a 75% ethanol aqueous solution for 60 seconds in a sterile laminar flow hood. Rinse 2-3 times with sterile water, then soak in a 2% NaClO aqueous solution for 15 minutes with continuous shaking. Finally, rinse 3-4 times with sterile water, 5 seconds each time. After the above disinfection treatment, inoculate the seeds into germination medium (MS medium containing 0.3 mg / L 6-BA and 0.9 mg / L NAA, with 0, 8.6, 17.2, 25.8, or 34.4 mg / L ZnSO4 added, adjusted to pH 5.8) for aseptic germination and culture to obtain complete *Limonium sinense* seedlings. After seed inoculation, the culture medium was placed under conditions of 2000 lx light intensity, 18 hours / day light duration, and 25 ± 1℃ for 30 days. Sixty bottles were prepared for each concentration, with one *Limonium sinense* seed inoculated into each bottle. Three biological replicates were set up for each concentration, and the germination rate and average plant height for each concentration were recorded on day 30 after 30 days.

[0034] Table 1. Effects of germination media with different concentrations of ZnSO4 on the germination and growth of *Limonium sibiricum* seeds.

[0035] 0 45.74±2.24d 5.09 ± 0.35 bc 8.6 98.52±1.19a 8.52±0.17a 17.2 84.81±3.90b 6.23±0.46b 25.8 63.52±2.86c 4.12±0.51c 34.4 44.44±5.85d 2.45±0.34d

[0036] Note: The data in the table are mean ± standard deviation. The letters after the data indicate the results of the significance analysis. Different letters indicate that there are significant differences between different treatments (p≦0.05).

[0037] Table 1 shows the germination effect of *Limonium sibiricum* seeds when inoculated into germination media supplemented with different concentrations of ZnSO4 (0, 8.6, 17.2, 25.8, 34.4 mg / L ZnSO4). The results in Table 1 indicate that the germination medium supplemented with 8.6 mg / L ZnSO4 had the most significant promoting effect on the germination of *Limonium sibiricum* seeds, significantly higher than other treatment groups, with an average germination rate of 98.52% (maximum) and an average plant height of 8.52 cm (maximum) on day 30. (Based on Table 1 and...) Figure 1 The germination rate and average plant height showed a trend of increasing ZnSO4 concentration from 0 mg / L to 8.6 mg / L. However, when the ZnSO4 concentration exceeded 8.6 mg / L, these values ​​significantly decreased with further increases in concentration. In conclusion, the germination medium supplemented with 8.6 mg / L ZnSO4 significantly promoted the germination of *Limonium sinense* seeds.

[0038] Example 2: Effect of adventitious bud induction medium containing different concentrations of CuSO4 on the effect of inducing adventitious buds on petioles of Limonium sibiricum

[0039] To investigate the effect of different concentrations of CuSO4 added to adventitious shoot induction media (MS medium containing 0.3 mg / L 6-BA and 0.9 mg / L NAA, with additional additions of 0, 0.16, 0.32, 0.64, 12.8, and 2.56 mg / L CuSO4, pH adjusted to 5.8) on the induction of adventitious shoots from petioles of *Limonium sibiricum*, six bottles of each concentration of induction medium were prepared, and two *Limonium sibiricum* petioles were inoculated into each bottle. Under a clean bench, the petioles of *Limonium sibiricum*, approximately 1.0 cm in length, were used as explants. These petioles were cut from the bud point (the bud at the junction of the petiole and stem segment) and laid horizontally in the adventitious shoot induction medium, ensuring the explants were embedded 2-3 mm below the surface of the medium to allow for sufficient contact between the petiole wound and the medium. The explants remained biologically active at the time of inoculation. Three biological replicates were set up for each concentration. The plants were cultured for 18 days at a room temperature of 25°C, a light intensity of 2000 lx, and a light duration of 12 hours per day. The callus induction rate, adventitious bud regeneration rate, and number of buds of the petiole explants were counted.

[0040] Table 2. Effects of adventitious bud induction medium with different concentrations of CuSO4 on the induction of adventitious buds from petioles of Limonium sibiricum.

[0041] 0 69.44±4.18b 27.23±1.31c 4.01±0.89c 0.16 97.22±1.72a 45.67±1.52b 10.33 ± 1.07 ab 0.32 99.45±0.43a 53.24±1.63a 14.08±1.23a 0.64 67.28±5.72b 43.12±2.08b 8.12±0.96b 1.28 66.67±4.45b 29.17±1.36c 4.67±1.15c 2.56 47.22±5.07c 25.20±1.46c 3.58±1.75c

[0042] Note: The data in the table are mean ± standard deviation. The letters after the data indicate the results of the significance analysis. Different letters indicate that there are significant differences between different treatments (p≦0.05).

[0043] From Table 2 and Figure 2 The results showed that inoculating *Limonium sibiricum* petiole explants into adventitious shoot induction media supplemented with different concentrations of CuSO4 (0, 0.16, 0.32, 0.64, 1.28, 2.56 mg / L CuSO4) for 18 days significantly promoted adventitious shoot differentiation. The results indicated that the adventitious shoot induction medium supplemented with 0.32 mg / L CuSO4 had the most significant promoting effect on adventitious shoot induction in *Limonium sibiricum*, achieving a callus induction rate of 99.45% (maximum), an adventitious shoot regeneration rate of 53.24% (maximum), and an average of 14.08 adventitious shoots per explant segment (maximum).

[0044] Example 3: Effect of adventitious bud induction medium with different concentrations of ZnSO4 on the effect of inducing adventitious buds on petioles of Limonium sibiricum

[0045] To investigate the effect of different concentrations of ZnSO4 added to adventitious shoot induction media (MS medium containing 0.3 mg / L 6-BA and 0.9 mg / L NAA, with ZnSO4 added at concentrations of 0, 0.13, 0.26, 0.52, 1.04, and 2.08 mg / L, adjusted to pH 5.8) on the induction of adventitious shoots from petioles of *Limonium sibiricum*, 30 bottles were prepared for each concentration, and two *Limonium sibiricum* petioles were inoculated into each bottle. Under a clean bench, petioles of *Limonium sibiricum*, approximately 1.0 cm in length, were used as explants. These petioles were cut from the stem segment (with the bud point removed) and laid horizontally in the adventitious shoot induction medium, ensuring the explants were embedded 2-3 mm below the surface of the medium to allow for sufficient contact between the petiole wound and the medium. The explants remained biologically active at the time of inoculation. Three biological replicates were set up for each concentration. The plants were cultured for 18 days at a room temperature of 25°C, a light intensity of 2000 lx, and a light duration of 12 hours per day. The callus induction rate, adventitious bud regeneration rate, and number of buds of the petiole explants were counted.

[0046] Table 3. Effects of adventitious bud induction media with different concentrations of ZnSO4 on the induction of adventitious buds from petioles of *Limonium sibiricum*.

[0047] 0 67.37±3.87b 26.48±1.29c 4.32±1.15c 0.13 95.75±2.34a 46.72±1.48b 6.58 ± 0.57 bc 0.26 98.67±0.68a 56.64±1.52a 13.75±2.64a 0.52 70.36±4.29b 44.53±2.12b 8.67±1.15b 1.04 68.25±4.68b 28.37±1.46c 8.02±1.09b 2.08 46.38±5.24c 24.34±2.58c 4.24±1.05c

[0048] Note: The data in the table are mean ± standard deviation. The letters after the data indicate the results of the significance analysis. Different letters indicate that there are significant differences between different treatments (p≦0.05).

[0049] Table 3 shows the effect of adding different concentrations of ZnSO4 (0, 0.13, 0.26, 0.52, 1.04, 2.08 mg / L ZnSO4) to the adventitious shoot induction medium for culturing *Limonium sibiricum* petiole explants for 18 days to induce adventitious shoot differentiation. The results indicate that the adventitious shoot induction medium supplemented with 0.26 mg / L ZnSO4 significantly improves the induction of adventitious shoots in *Limonium sibiricum*. Figure 3 Among them, A) had the most obvious promoting effect, with a callus rate of 98.67% (maximum), an adventitious bud regeneration rate of 56.64% (maximum), and an average number of adventitious buds per explant segment of 13.75 (maximum).

[0050] Example 4: Effect of adventitious bud induction medium with different concentrations of AgNO3 on the effect of inducing adventitious buds on petioles of Limonium sibiricum

[0051] To investigate the effects of adding different concentrations of AgNO3 to adventitious shoot induction media (MS medium containing 0.3 mg / L 6-BA and 0.9 mg / L NAA, with 0, 0.14, 0.42, 1.00, 1.57, and 3.15 mg / L AgNO3 added respectively), 30 bottles were prepared for each concentration, and two petioles of *Limonium sibiricum* were inoculated into each bottle. On a clean bench, petioles of *Limonium sibiricum* were used as explants. The petioles were small segments approximately 1.0 cm in length, with the bud point (the bud at the junction of the petiole and stem segment) removed. These explants were laid horizontally in the adventitious shoot induction medium, 2-3 mm below the surface of the medium to ensure sufficient contact between the petiole wound and the medium. The explants remained biologically active at the time of inoculation. Three biological replicates were set up for each concentration. The plants were cultured for 18 days at a room temperature of 25°C, a light intensity of 2000 lx, and a light duration of 12 hours per day. The callus induction rate, adventitious bud regeneration rate, and number of buds of the petiole explants were counted.

[0052] Table 4. Effects of adventitious shoot induction medium with different concentrations of AgNO3 on the induction of adventitious shoots from petioles of *Limonium sibiricum*.

[0053] 0 69.44±4.83c 27.39±1.37c 4.38±0.57c 0.14 90.56±3.62a 50.28±1.42a 12.32±1.13a 0.42 84.76 ± 4.65 ab 45.36±1.39b 7.33 ± 1.15 bc 1 80.06±1.73b 43.21±2.45b 5.69±2.08c 1.57 78.08 ± 4.8 bc 33.72±1.64c 2.83±0.39d 3.15 73.51±4.58c 29.53±2.48c 2.67±0.52d

[0054] Note: The data in the table are mean ± standard deviation. The letters after the data indicate the results of the significance analysis. Different letters indicate that there are significant differences between different treatments (p≦0.05).

[0055] Table 4 shows the effects of adding different concentrations of AgNO3 (0, 0.14, 0.42, 1.00, 1.57, and 3.15 mg / L AgNO3) to MS medium on the induction of adventitious shoots from *Limonium sibiricum* petiole explants for 18 days. The results indicate that the adventitious shoot induction medium with an additional 0.14 mg / L AgNO3 significantly improved the induction of adventitious shoots in *Limonium sibiricum*. Figure 3 Among them, B) had the most obvious promoting effect, with a callus rate of 90.56% (maximum), an adventitious bud regeneration rate of 50.28% (maximum), and an average number of adventitious buds per explant segment of 12.32 (maximum).

Claims

1. The application of a germination culture medium in the germination of *Limonium stenoptera* seeds, characterized in that, The germination medium consists of the following components: 0.3 mg / L 6-BA, 0.9 mg / L NAA, 8.6 mg / L ZnSO4, with the remainder being MS medium.

2. The application of an adventitious bud induction medium in inducing adventitious buds on the petioles of *Limonium sibiricum*, characterized in that... The composition of the adventitious bud induction medium is any one of the following (1)-(3): (1) 0.3 mg / L 6-BA, 0.9 mg / L NAA, 0.32 mg / L CuSO4, with the remainder being MS medium; (2) 0.3 mg / L 6-BA, 0.9 mg / L NAA, 0.26 mg / L ZnSO4, with the remainder being MS medium; (3) 0.3 mg / L 6-BA, 0.9 mg / L NAA, 0.14 mg / L AgNO3, with the remainder being MS medium.

3. A method for inducing the differentiation of *Limonium sibiricum* into explants, characterized in that, Includes the following steps: S1. After sterilization, the seeds of *Limonium sinense* were inoculated into a germination medium and cultured for 30 days to obtain sterile seedlings. The germination medium consisted of the following: 0.3 mg / L 6-BA, 0.9 mg / L NAA, 8.6 mg / L ZnSO4, with the remainder being MS medium. S2. Take the petioles of the sterile seedlings obtained in step S1 as explants and inoculate them into the adventitious bud induction medium to induce adventitious buds; The composition of the adventitious bud induction medium is any one of the following (1)-(3): (1) 0.3 mg / L 6-BA, 0.9 mg / L NAA, 0.32 mg / L CuSO4, with the remainder being MS medium; (2) 0.3 mg / L 6-BA, 0.9 mg / L NAA, 0.26 mg / L ZnSO4, with the remainder being MS medium; (3) 0.3 mg / L 6-BA, 0.9 mg / L NAA, 0.14 mg / L AgNO3, with the remainder being MS medium.

4. The method according to claim 3, characterized in that, The seeds mentioned in step S1 are cone-shaped, plump seeds without obvious lesions or mold.

5. The method according to claim 3, characterized in that, The disinfection process described in step S1 is as follows: soak the seeds in a 75% ethanol aqueous solution for 60 seconds, rinse them 2-3 times with sterile water, then soak them in a 2% NaClO aqueous solution for 15 minutes with continuous shaking, and finally rinse them 3-4 times with sterile water for 5 seconds each time.

6. The method according to claim 3, characterized in that, The petiole mentioned in step S2 is a petiole segment with a length of 1 cm after removing the bud point at the connection between the petiole and the stem segment.

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