Method for directly inducing whole-plant regeneration of limonium aureum

Abstract

The invention discloses a method for directly inducing whole-plant regeneration of limonium aureum. The method comprises the following steps: segmenting the petioles of sterile limonium aureum seedlings cultivated for 30 days, respectively soaking the petioles in high-concentration NAA, 6-BA and IBA solutions for 20-30 minutes, and then transferring the petioles into an MS culture medium to carry out induction culture for 48 days, so as to obtain regenerated complete plants differentiated from the petioles. According to the method disclosed by the invention, additional steps of callus induction, adventitious bud elongation culture, adventitious bud rooting culture and the like are not needed, a regenerated complete plant with a root can be directly obtained from explant induction, and a set of efficient solution is provided for the rapid propagation problem of the limonium aureum; the problems that aseptic seedlings are difficult to obtain through tissue culture, the tissue culture period is long, subculture and updating culture procedures are tedious, and materials are prone to pollution are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cell engineering, and in particular relates to a method for directly inducing the whole plant regeneration of Limonium xanthophyllum. Background Art

[0002] Limonium chinense, also known as golden spoon grass, yellow rock pine, and golden limonium, is a perennial herbaceous plant of the genus Limonium in the family Pleurotus eryngii. Limonium chinense is a salt-alkali-tolerant and drought-tolerant plant that often grows in arid environments such as the Gobi Desert, tidal flats, and mobile sand dunes. It is mostly distributed in Xinjiang, Gansu, Shaanxi, Qinghai, and Inner Mongolia in my country. Limonium chinense has extremely high medicinal value and has the effects of anti-inflammatory and hemostasis, regulating menstruation and benefiting the spleen, nourishing blood and anti-cancer, and treating pain. Its roots can be used to relieve symptoms of weakness and loss of appetite, its leaves can help treat gastric ulcers and gastric cancer, and its flowers and branches can be used to treat functional uterine bleeding, cervical cancer, and other types of bleeding.

[0003] Yellow Limonium has a high ornamental value. Its long flowering period, bright colors, and short plants make it very suitable as an administrative ornamental flower. The membranous and tough petals allow it to maintain a long-lasting bright color, making it a good material for making cut flowers and dried flowers. Wild Yellow Limonium sprouts year after year, is evergreen, has strong adaptability to desert arid environments, and grows in concentrated areas. It is an excellent source of feed for the arid grasslands of the northwest.

[0004] At present, the main method of reproduction of Limonium yellowii is seed reproduction, but the seeds of Limonium yellowii are small, the maturity period is unstable, the annual yield is low, and seed reproduction is difficult; while the traditional tissue culture technology requires several steps such as sterile seedling cultivation, callus induction, adventitious bud differentiation, and rooting culture. It has many processes, a culture cycle of up to 120-150 days, and is prone to contamination, which seriously hinders the development of tissue culture technology of Limonium yellowii. Summary of the invention

[0005] The purpose of the present invention is to provide a method for rapidly and directly inducing Limonium candidum explants to obtain regenerated complete plants in response to the problems of existing Limonium candidum tissue culture technology that is too time-consuming, has too many subculture steps, and is prone to contamination.

[0006] The invention adopts a high concentration hormone solution (40 mg / L NAA, 30 mg / L 6-BA, 10 mg / L IBA solution) to promote efficient differentiation and rooting of adventitious buds of Limonium candidum explants. The petiole explants of Limonium candidum are soaked in the high concentration hormone solution, and then the explants are inoculated into MS culture medium without hormone addition for 48 days to obtain complete plants with a plant height of 5-11 cm and a root length of more than 6 cm.

[0007] The object of the present invention is to provide a method for directly inducing the regeneration of Limonium candidum plants, comprising the following steps:

[0008] S1. Take the petioles of sterile Limonium luteum seedlings and cut them into small pieces as explants;

[0009] S2. After soaking the explant in the hormone solution for 20-30 minutes, take it out and dry the residual solution on the surface of the explant;

[0010] S3. The explants treated in step S2 are inoculated into MS medium for whole plant regeneration induction culture;

[0011] The hormone solution is 40 mg / L NAA solution, 30 mg / L 6-BA solution or 10 mg / L IBA solution.

[0012] Preferably, the sterile seedlings of Limonium candidum are obtained by the following method: selecting Limonium candidum seeds, washing and disinfecting them with distilled water, soaking them in 100 μg / L GA3 solution for 2 hours, and then inoculating them into MS culture medium for sterile seedling development culture.

[0013] Preferably, the small segment is 1.0-2.0 cm long.

[0014] Preferably, the immersion of the explant in the hormone solution for 20-30 min is immersion of the explant in a 40 mg / L NAA solution for 20 min, immersion of the explant in a 30 mg / L 6-BA solution for 30 min, or immersion of the explant in a 10 mg / L IBA solution for 30 min.

[0015] Preferably, the MS medium is composed of: 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 copper sulfate pentahydrate, 0.025mg / L cobalt chloride hexahydrate, 37.3mg / L sodium ethylenediaminetetraacetate dihydrate, 27.8mg / L ferrous sulfate heptahydrate, 2mg / L glycine, 0.1mg / L thiamine hydrochloride, 0.5mg / L pyridoxine hydrochloride, 0.5mg / L niacin, 100mg / L inositol, 30g / L sucrose and 6g / L agar, the balance is water, and the pH is adjusted to 5.8-6.0.

[0016] Preferably, the culture conditions are light intensity 2000 lx, light time 12 hours / day, and temperature 25±1°C.

[0017] Preferably, the Limonium xanthophyllum seeds are healthy, plump and round Limonium xanthophyllum seeds harvested within two years.

[0018] Preferably, the cleaning with distilled water is: using flowing distilled water to rinse impurities on the surface of the seeds for 3 minutes, and then transferring to sterile conditions and rinsing with sterile distilled water for 1 minute.

[0019] Preferably, the disinfection is as follows: the Limonium candidum seeds are soaked in a 75% ethanol aqueous solution by volume for 30 seconds, then soaked in a 2% NaClO aqueous solution by mass for 15 minutes, and rinsed with sterile distilled water 2-3 times, each time for 30 seconds to 60 seconds; during the soaking period, the Limonium candidum seeds need to be continuously dispersed to ensure thorough disinfection.

[0020] Preferably, the time for culturing the sterile seedlings is 30 days.

[0021] Beneficial effects of the present invention:

[0022] The present invention mainly changes the traditional method of tissue culture of Limonium candidum and simplifies the tissue culture steps, that is, using a high concentration of plant hormone solution (40 mg / L NAA, 30 mg / L 6-BA, 10 mg / L IBA solution) to soak the petioles of Limonium candidum, thereby stimulating the petioles to quickly absorb the plant hormones, inducing tissue cells to more efficiently and quickly dedifferentiate to produce adventitious buds and root systems, and then develop into complete regenerated plants.

[0023] By using the present invention, it takes about 78 days from inoculating yellow flower Limonium seeds to cultivating sterile seedlings, obtaining sterile explants, inoculating to differentiation and developing into complete Limonium plants, of which explant differentiation only takes about 48 days to form a regenerated complete plant, which can save 42-72 days compared with the traditional tissue culture method of adding hormones to the culture medium to induce callus tissue. In addition, since the method has simple operation steps, is convenient and fast, and has low cost, it can provide development possibilities for the mechanized production and automated production of yellow flower Limonium tissue culture technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the effect of whole-plant regeneration induction culture after petiole explants of Limonium candidum were immersed in high-concentration hormone solution; A and B are the effects of petiole explants of Limonium candidum immersed in 40 mg / L NAA solution for 20 min and then inoculated on MS medium and cultured for 48 days; C and D are the effects of petiole explants of Limonium candidum immersed in 30 mg / L 6-BA solution for 30 min and then inoculated on MS medium and cultured for 48 days; E and F are the effects of petiole explants of Limonium candidum immersed in 10 mg / L IBA solution for 30 min and then inoculated on MS medium and cultured for 48 days; bars=1.0 cm.

[0025] Figure 2is the effect of whole-plant regeneration induction culture of Limonium candidum petiole explants using the traditional method (directly inoculated into hormone-added culture medium without soaking in hormone solution); A is the effect of Limonium candidum petiole explants directly inoculated into MS culture medium without soaking in hormone solution and cultured for 48 days (control group), B is the effect of Limonium candidum petiole explants directly inoculated into MS culture medium supplemented with 0.4 mg / L NAA without soaking in hormone solution and cultured for 48 days, C is the effect of Limonium candidum petiole explants directly inoculated into MS culture medium supplemented with 0.3 mg / L 6-BA without soaking in hormone solution and cultured for 48 days, D is the effect of Limonium candidum petiole explants directly inoculated into MS culture medium supplemented with 0.5 mg / L IBA without soaking in hormone solution and cultured for 48 days; bars=1.0 cm. DETAILED DESCRIPTION

[0026] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0027] The MS medium used in the following examples has the following formula: 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 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 and 6 g / L agar, with the balance being water. Specifically, the MS culture medium was adjusted to pH 5.8-6.0 using 1 mol / L HCl solution or 1 mg / L NaOH solution, and then sterilized by high pressure at 121° C. and 0.1 MPa for 20 min.

[0028] The culture conditions of the sterile seedling development and whole plant regeneration induction culture stages of Limonium chrysantha were 2000 lx light intensity, 12 hours / day light duration, and 25 ± 1 ° C culture temperature. All experimental treatments were repeated three times, and the experimental data were expressed as the mean ± standard deviation of 3 repeated experiments. All data were analyzed by SPSS Statistics 26.0 statistical analysis software for variance analysis and Duncan multiple comparisons (P ≦ 0.05). Different letters after the data indicate significant differences between treatments.

[0029] Example 1: Obtaining Limonium candidum explants

[0030] (1) Selection, cleaning and disinfection of Limonium truncatum seeds

[0031] Select an appropriate amount of healthy, plump, and round seeds harvested within the past two years. Use running distilled water to rinse the impurities on the surface of the seeds for 3 minutes under conventional sterile conditions. Transfer the seeds to a clean bench in a sterile environment and rinse the seeds 2-3 times with sterilized distilled water, each time for 1 minute. Then soak the seeds in a 75% ethanol aqueous solution for 30 seconds, and soak the seeds in a 2% NaClO aqueous solution for 15 minutes. During this period, you need to shake them constantly to break up the seeds that have clumps together to prevent the seeds from agglomerating and causing incomplete disinfection. Finally, rinse the seeds 2-3 times with running distilled water, each time for 30-60 seconds.

[0032] (2) Cultivation of aseptic seedlings of Limonium scabra

[0033] Prepare 100μg / L GA3 solution in advance before soaking the seeds of Limonium chrysantha: dissolve an appropriate amount of GA3 powder in a small amount of anhydrous ethanol, add distilled water to a concentration of 100μg / L after complete dissolution, adjust the pH of the solution to 5.8-6.0, and filter and sterilize the prepared 100μg / L GA3 solution with a 0.22 micron water filter membrane (Millipore, USA) before use. Soak the washed Limonium chrysantha seeds in the 100μg / L GA3 solution for 2h, shaking the seeds every half an hour. Use sterile absorbent paper to absorb the surface moisture of the soaked seeds, and inoculate the seeds into MS culture medium without hormone addition and solidified and cooled. Inoculate 2 seeds per bottle of culture medium, and culture for 30d at room temperature 25±1℃, light 2000lx, and light time 12 hours / day to obtain sterile seedlings of Limonium chrysantha.

[0034] (3) Preparation of plant hormone solution and acquisition of Limonium scabra explants

[0035] Accurately weigh a certain amount of naphthaleneacetic acid (NAA), 6-benzylaminopurine (6-BA), and 3-indolebutyric acid (IBA) powders, fully dissolve NAA and 6-BA with 1 mol / L NaOH solution, fully dissolve IBA with anhydrous ethanol, and then dilute with deionized water to prepare different concentrations of NAA (0, 20, 40, 80 and 120 mg / L) solutions, 6-BA (0, 15, 30, 45 and 90 mg / L) solutions, and IBA (0, 5, 10, 20 and 40 mg / L) solutions. Before use, adjust the pH of the solution to 5.8-6.0, and filter and sterilize the prepared NAA, 6-BA, and IBA solutions with a 0.22 micron water filter membrane (Millipore, USA). The yellow flower Limonium explants are obtained from the sterile seedlings cultivated in steps (1)-(2). Select sterile seedlings with vigorous growth, upright plants, emerald green leaves, thick leaves, spindle-shaped leaves, 9-14 leaves, white and tender roots, and only the connection between the root base and the bottom petiole turns red. Take the leaves with better growth in the sterile seedlings under the sterile environment of the clean bench, cut off the petiole from the connection between the petiole and the main stem, remove the leaf part, and only keep the petiole. Further cut the petiole into small pieces of 1.0-2.0cm long as explants for further experiments.

[0036] Example 2: Soaking of Limonium candidum explants and whole plant regeneration induction culture

[0037] The petiole segments obtained in Example 1 were immersed in 6-BA, NAA, and IBA solutions of different concentrations for 20-30 minutes, during which the hormone solutions were gently shaken. After the soaking, all the explants were removed using tweezers and the hormone solution remaining in the petioles was absorbed using sterile absorbent paper. The explants were placed horizontally and gently sunk into the surface of the culture medium, and cultured for 48 days at a light intensity of 2000 lx, a light duration of 12 hours / day, and a temperature of 25±1°C.

[0038] After 48 days of cultivation, the number of callus explants, callus induction rate (the percentage of callus explants to the total number of explants), the number of seedling explants (plant individuals with complete above-ground and underground parts differentiated from explants), the seedling induction rate (the percentage of explants that formed complete plants to the total number of explants), the average plant height (plant height is the vertical height from the junction of the differentiated seedlings and the original explant to the highest point where the leaves at the top of the plant naturally extend, and the average plant height is the average height of all differentiated seedlings after 48 days of cultivation), and the average taproot length is the average length of the taproots of all differentiated seedlings.

[0039] The details are as follows:

[0040] (1) Effect of soaking in NAA solution of different concentrations on petiole regeneration of Limonium fulva

[0041] The petiole explants of Limonium fulgens obtained in Example 1 were soaked in NAA solutions of different concentrations (0, 20, 40, 80 and 120 mg / L) for 20 minutes and inoculated into MS medium. Ten bottles were inoculated at each concentration, and two explants were inoculated in each bottle; each concentration was repeated three times, and statistical data was collected and analyzed after 48 days of culture.

[0042] Table 1 Effects of soaking in NAA solution of different concentrations for 20 min on petiole differentiation of Limonium fulva

[0043]

[0044] Note: The letters after the data indicate the results of significance analysis, and different letters indicate significant differences between different treatments (p<0.05); the concentration of 0 mg / L means no hormone solution was added, and the solution was immersed in sterile distilled water as the control group.

[0045] From Table 1, it can be analyzed that when the petioles of Limonium candidum were immersed in 0-120 mg / L NAA solution for 20 minutes, the immersion in 40 mg / L NAA solution had the best effect on promoting the differentiation and growth of the petioles of Limonium candidum, and the highest callus induction rate of 91.68% was obtained. The number of seedling explants was the largest, the seedling rate was as high as 78.35%, and the growth effect was also the most significant among the same treatments. The average plant height could reach 10.80 cm, and the average taproot length could reach 8.34 cm.

[0046] (2) Effects of soaking in NAA solution of the same concentration for different time periods on petiole regeneration of Limonium fulva

[0047] Furthermore, different soaking times significantly affect the absorption of NAA by the explants.

[0048] The petioles of grass were immersed for different time periods (0, 10, 20, 50, 100 min) and inoculated into MS medium for 48 days. Ten bottles were inoculated for each treatment time, with two explants in each bottle; each treatment time was repeated three times, and the data were collected and analyzed after 48 days of inoculation.

[0049] Table 2 Effects of soaking in 40 mg / L NAA solution for different time periods on petiole differentiation of Limonium fulva

[0050]

[0051] Note: The letters after the data indicate the results of significance analysis, and different letters indicate significant differences among different treatments (p<0.05); the 0 min soaking treatment means that the explants were not soaked and were directly inoculated into the culture medium, which served as the control group.

[0052] From Table 2, it can be seen that when the petiole explants are soaked in 40mg / L NAA solution, as the soaking time increases, the callus induction rate of the petiole increases significantly. When the soaking time is 20min, the induction rate of the petiole reaches the maximum value, averaging 98.34%, and the seedling rate also reaches the maximum value, averaging 76.68%. Under this soaking time treatment, the growth and differentiation effect of the explants is excellent, the average plant height can reach 10.42cm, and the average taproot length can reach 10.2cm. Further analysis shows that when the soaking time is gradually extended, the differentiation effect of the petiole gradually decreases. The analysis shows that 20min is the best soaking time for 40mg / L NAA solution.

[0053] (3) Effects of soaking in 6-BA solutions of different concentrations on petiole regeneration of Limonium fulva

[0054] The petioles of Limonium fulgens obtained in the example were soaked in 6-BA solutions of different concentrations (0, 15, 30, 45, 90 mg / L) for 30 minutes, inoculated into MS medium and cultured for 48 days. Ten bottles were inoculated at each concentration, and two explants were inoculated in each bottle; each concentration was repeated 3 times, and after inoculation, the data were counted and analyzed for 48 days.

[0055] Table 3 Effects of immersion in 6-BA solution of different concentrations for 30 min on petiole differentiation of Limonium fulvatum

[0056]

[0057]

[0058] Note: The letters after the data indicate the results of significance analysis, and different letters indicate significant differences between different treatments (p<0.05); the concentration of 0 mg / L means no hormone solution was added, and the solution was immersed in sterile distilled water as the control group.

[0059] From Table 3, it can be analyzed that when the petioles of Limonium candidum were immersed in 0-90 mg / L 6-BA solution for 30 minutes, the experimental results showed that immersion in 30 mg / L 6-BA solution had the best effect on promoting the differentiation and growth of Limonium candidum petioles, and the highest callus induction rate of 81.64% was obtained, the number of seedling explants was the largest, the seedling rate was as high as 78.31%, and the growth effect was also the most significant among the same treatments, with an average plant height of 8.61 cm and an average taproot length of 10.10 cm.

[0060] (4) Effects of soaking in 6-BA solution of the same concentration for different time periods on petiole regeneration of Limonium fulva

[0061] Furthermore, the effect of the soaking time on the differentiation effect of the explants absorbing the plant hormone 6-BA was explored. The petioles of the yellow flowered Limonium obtained in Example 1 were soaked for different times (0, 15, 30, 45, 90 min) using the preferred 30 mg / L 6-BA solution, and then inoculated into MS medium for 48 days of culture. Ten bottles were inoculated for each treatment time, with two explants inoculated in each bottle; each time treatment was repeated 3 times, and statistical data was collected and analyzed after 48 days of culture after inoculation.

[0062] Table 4 Effects of immersion in 30 mg / L 6-BA solution for different time periods on petiole differentiation of Limonium fulva

[0063]

[0064] Note: The letters after the data indicate the results of significance analysis, and different letters indicate significant differences among different treatments (p<0.05); the 0 min soaking treatment means that the explants were not soaked and were directly inoculated into the culture medium, which served as the control group.

[0065] It can be seen from Table 4 that when the petiole explants were soaked in 30mg / L 6-BA solution, the callus induction rate of the petiole increased significantly with the increase of soaking time. When the soaking time was 30min, the induction rate of the petiole reached the maximum value, averaging 86.65%, and the seedling rate also reached the maximum value, averaging 78.62%. Under this soaking time treatment, the growth and differentiation effect of the explants was excellent, the average plant height could reach 8.92cm, and the average taproot length could reach 10.21cm. Further analysis showed that when the soaking time was gradually extended, the differentiation effect of the petiole gradually decreased. The analysis showed that 30min was the best soaking time for 30mg / L 6-BA solution.

[0066] (5) Effect of soaking in IBA solution of different concentrations on petiole regeneration of Limonium fulva

[0067] The petioles of Limonium fulgens obtained in Example 1 were soaked in IBA solutions of different concentrations (0, 5, 10, 20, 40 mg / L) for 30 minutes, inoculated into MS medium and cultured for 48 days. Ten bottles were inoculated at each concentration, and two explants were inoculated in each bottle; each concentration was repeated three times, and statistical data was collected and analyzed after 48 days of culture after inoculation.

[0068] Table 5 Effects of soaking in IBA solution of different concentrations for 30 min on petiole differentiation of Limonium fulva

[0069]

[0070] Note: The letters after the data indicate the results of significance analysis, and different letters indicate significant differences between different treatments (p<0.05); the concentration of 0 mg / L means no hormone solution added, and the sterile distilled water immersion treatment was used as the control group.

[0071] From the data analysis in Table 5, it can be seen that when the treatment concentration reaches 10 mg / L, the differentiation effect of Limonium fulgens is the best, and the highest average of 18.68 callus explants and 14.63 seedling individuals can be obtained, and the seedling rate is as high as 73.34%. The growth effect of the regenerated plants is the best, and the average plant height of 9.15 cm and the average taproot length of 10.13 cm can be obtained on the 48th day. As the concentration of the immersion treatment increases, the differentiation effect of the explants decreases significantly.

[0072] (6) Effects of soaking in IBA solution of the same concentration for different time periods on petiole regeneration of Limonium fulva

[0073] In order to explore the effect of different soaking times on the regeneration of petioles of Limonium chrysantha, petioles were treated with different soaking times at the preferred 10 mg / L IBA concentration. The petioles of Limonium chrysantha were soaked in 10 mg / L IBA solution for different times (0, 15, 30, 45, 90 min), inoculated into MS medium and cultured for 48 days. Ten bottles were inoculated for each treatment time, and two explants were inoculated in each bottle; each time treatment was repeated 3 times, and the statistics were collected and analyzed after 48 days of culture after inoculation.

[0074] Table 6 Effects of soaking in 10 mg / L IBA solution for different time periods on petiole differentiation of Limonium fulva

[0075]

[0076]

[0077] Note: The letters after the data indicate the results of significance analysis, and different letters indicate significant differences among different treatments (p<0.05); the 0 min soaking treatment means that the explants were not soaked and were directly inoculated into the culture medium, which served as the control group.

[0078] From Table 6, it can be seen that as the soaking time increases, when the soaking time of 10mg / L IBA solution reaches 30min, the petiole differentiation effect of Limonium fulgens is the best, the callus explants can reach a maximum of 17.61, and the induction rate is as high as 88.32%; the seedling effect is the best, reaching 14.64, and the seedling rate reaches 73.38%. Under this soaking time treatment, the plants differentiated from the explants can reach a maximum average plant height of 9.41cm and an average taproot length of 11.26cm. Based on the above, 30min is preferably used as the soaking time of 10mg / L IBA solution.

[0079] Example 3: Effects of adding different hormones directly into the culture medium on the regeneration of Limonium candidum petioles

[0080] In order to compare the effects of the traditional method (directly adding different types of hormones to the culture medium) and the soaking treatment method on the regeneration of the petiole of Limonium chrysantha, it is preferred to use different types of hormones (0 (control, no hormone added), 0.4 mg / L NAA, 0.3 mg / L6-BA, 0.5 mg / L IBA, the selected hormone concentration is a lower concentration of hormone commonly used to induce explant regeneration) and add them to the MS culture medium respectively, wherein the group without any hormone treatment is used as the control group. The petiole of Limonium chrysantha obtained in Example 1 was segmented and directly inoculated into the MS culture medium containing the above-mentioned different types of hormones for culture without soaking treatment; 10 bottles were inoculated for each hormone treatment, and 2 segments of explants were inoculated in each bottle; each concentration was repeated 3 times, and the number of callus explants, callus induction rate, number of seedling explants and seedling rate of the petiole of Limonium chrysantha were counted and analyzed after 48 days of culture.

[0081] Table 7 Effects of adding different hormones directly to MS medium on petiole regeneration of Limonium fulva

[0082] combination Number of callus explants Callus induction rate (%) Number of explants grown into seedlings Seedling rate (%) 0 3.68±0.62a 18.31±3.32a 0 0 0.4mg / L NAA 4.32±0.31a 21.64±1.64a 0 0 0.3mg / L 6-BA 3.39±0.37a 16.58±1.58a 0 0 0.5mg / L IBA 1.28±0.34b 6.67±1.65b 0 0

[0083] Note: The letters after the data represent the results of significance analysis. Different letters indicate significant differences between different treatments (p<0.05); 0 means no hormones were added to the MS medium, which served as the control group.

[0084] From the analysis of Table 7, it can be seen that the regeneration effects of 0.4mg / L NAA, 0.3mg / L 6-BA, 0.5mg / L IBA and the control group of Limonium chrysantha were not ideal, only a few explants produced callus tissue, the number of explants was 1.28-4.32, and the callus induction rate was 6.67%-21.64%. In addition, the addition of a single hormone cannot produce explants that can differentiate into complete plants. As can be seen from Table 7, the seedling rate of the traditional method of adding plant hormones is 0. Therefore, the callus tissue produced by the traditional method still needs to be followed by the regeneration induction of adventitious buds, the elongation culture of adventitious buds, and the rooting culture of adventitious buds to form a complete regenerated plant, and the steps are relatively cumbersome and the required time is long.

Claims

1. A method for directly inducing the regeneration of Limonium candidum plants, characterized in that: The following steps are involved: S1. Take the petioles of sterile Limonium luteum seedlings and cut them into small pieces as explants; S2. After soaking the explant in the hormone solution for 20-30 minutes, take it out and dry the residual solution on the surface of the explant; S3. The explants treated in step S2 are inoculated into MS medium for whole plant regeneration induction culture; The hormone solution is 40 mg / L NAA solution, 30 mg / L 6-BA solution or 10 mg / L IBA solution.

2. The method according to claim 1, characterized in that The sterile seedlings of Limonium truncatum are obtained by the following method: selecting Limonium truncatum seeds, washing and disinfecting them with distilled water, soaking them in 100 μg / L GA3 solution for 2 hours, and then inoculating them into MS culture medium for sterile seedling development.

3. The method according to claim 1, characterized in that The small segment is a small segment with a length of 1.0-2.0 cm.

4. The method according to claim 1, characterized in that: The explants are immersed in the hormone solution for 20-30 minutes, which means immersing the explants in 40 mg / L NAA solution for 20 minutes, immersing the explants in 30 mg / L 6-BA solution for 30 minutes, or immersing the explants in 10 mg / L IBA solution for 30 minutes.

5. The method according to claim 1, characterized in that The MS medium is composed of: 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 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 and 6 g / L agar, the balance is water, and the pH is adjusted to 5.8-6.

0.

6. The method according to claim 1, characterized in that The culture conditions are as follows: light intensity 2000 lx, light duration 12 hours / day, and temperature 25±1°C.

7. The method according to claim 2, characterized in that The yellow flower Limonium seeds are healthy, full and round yellow flower Limonium seeds harvested within two years.

8. The method according to claim 2, characterized in that: The distilled water cleaning is as follows: using flowing distilled water to rinse the impurities on the surface of the seeds for 3 minutes, and then transferring them to sterile conditions and rinsing them with sterile distilled water for 1 minute.

9. The method according to claim 2, characterized in that: The disinfection is as follows: the yellow flower Limonium seeds are soaked in a 75% ethanol aqueous solution by volume for 30 seconds, then soaked in a 2% NaClO aqueous solution by mass for 15 minutes, and rinsed with sterile distilled water for 2-3 times, each time for 30 seconds to 60 seconds; during the soaking period, the yellow flower Limonium seeds need to be continuously dispersed to ensure thorough disinfection.

10. The method according to claim 2, characterized in that The time for culturing the sterile seedlings is 30 days.

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