A method for rapid seedling cultivation of Eucommia ulmoides tissue culture

Abstract

This invention discloses a method for rapid seedling cultivation of Eucommia ulmoides via tissue culture, relating to the field of Eucommia ulmoides breeding technology. The method includes the following steps: using young, tender stem segments with axillary buds from superior Eucommia ulmoides strains as explants, after surface disinfection, the segments are cut into sections with axillary buds and inoculated into an axillary bud induction medium to induce axillary bud germination; the germinated axillary buds are cut into segments and inoculated into a specific medium for adventitious bud induction and elongation culture; the adventitious buds after proliferation culture are separated and subjected to a two-step in-bottle rooting method or external adventitious root induction, ultimately obtaining complete regenerated Eucommia ulmoides plants. Beneficial effects: This invention develops a simplified Eucommia ulmoides tissue culture propagation method that shortens the production cycle, has a high propagation coefficient, rapid rooting, high adventitious root induction rate, and minimal basal callus. It can rapidly obtain high-quality Eucommia ulmoides seedlings, which is of great significance for the large-scale propagation of superior strains and varietal improvement research, while also providing technical support for the sustainable development of the Eucommia ulmoides industry.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, specifically to a method for rapid seedling cultivation of Eucommia ulmoides via tissue culture. Background Technology

[0002] Eucommia ulmoides Oliver, belonging to the Eucommia genus of the Eucommiaceae family, is also known as silk-linked bark, silk cotton bark, cotton tree bark, and Chinese rubber tree because its bark and branches contain white fibrous strands when broken. It is a living fossil plant unique to China, with a cultivation history of nearly a thousand years. It is a precious tree species endemic to my country and has been designated as a national second-class protected plant. Eucommia has a wide range of uses. Its bark has been a valuable traditional Chinese medicine since ancient times and can also be planted as a street tree, shade tree, and in scenic forests. Eucommia is a treasure trove, possessing "three effects" (medicinal, therapeutic, and environmental benefits), and has extremely high development and utilization value. The bark contains active ingredients such as lignans, iridoids, phenylpropanoids, and terpenoids, which are widely used in the traditional Chinese medicine industry and have important medicinal and health-preserving effects. The abundant eucommia gum contained in the bark, leaves, and fruit of Eucommia ulmoides has the properties of acid resistance, alkali resistance, abrasion resistance, high insulation, and adhesion. It is one of the essential materials for manufacturing submarine cables, and is also suitable for the aerospace industry and the manufacture of electrical insulation equipment. It is a substitute resource for rubber trifoliate rubber, which has great development potential among the world's natural rubber resources.

[0003] Currently, in production, Eucommia ulmoides is mainly propagated by sowing. However, as a dioecious plant, seeds are prone to aging during storage, resulting in a short seed lifespan (0.5-1 year). Furthermore, Eucommia typically takes more than 10 years to flower and bear fruit; the seeds contain eucommia gum, requiring specific harvesting times and treatment to break dormancy. Therefore, seed propagation is cumbersome, time-consuming, and has a low germination rate. While traditional asexual propagation methods such as cuttings, grafting, and root layering have been reported for Eucommia propagation, they also suffer from cumbersome processes, time and seasonal limitations, and low efficiency.

[0004] Chinese patent application CN112931227A discloses a method for inducing plant regeneration from all parts of Eucommia ulmoides and constructing a transgenic plant regeneration system. The method includes the following steps: inducing callus tissue from Eucommia ulmoides embryos, stems, and / or roots; differentiating and culturing the callus tissue to obtain complete regenerated Eucommia ulmoides plants; the culture medium for differentiation culture is: basal medium + 0.5-2 mg / L 6-BA + 1.0-2.0 mg / L IAA + 25-35 g / L sucrose + 8-10 g / L agar, with a pH of 5.8-6.0. However, the rooting rate of the above formula is low (maximum 72%), and it takes about 20 days for roots to form. In our previous patent CN 113383706 A, we disclosed a method for efficient regeneration of Eucommia ulmoides based on LED light quality regulation. Although a large number of tissue culture seedlings were obtained through different light quality regulation and hormone ratios, there are still problems such as slow rooting, short root length, and large callus. Moreover, it is very costly to modify LED lights with different light qualities in tissue culture factories.

[0005] While research on the propagation of Eucommia ulmoides using plant tissue culture technology has been reported, it mainly focuses on using seedlings as explants. This approach suffers from problems such as severe pollution, low adventitious bud proliferation efficiency, and low rooting efficiency, seriously limiting the germplasm preservation and large-scale propagation of superior Eucommia ulmoides varieties. Eucommia ulmoides is a dioecious plant, requiring cross-pollination. If sexual propagation is used, the offspring cannot fully retain the superior genes of the parent plant, and the method is also limited by time and season. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to provide a method suitable for efficient regeneration and in vitro seedling formation of Eucommia ulmoides, with a high propagation coefficient, fast root emergence, and high adventitious root induction rate.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] A method for rapid seedling cultivation of Eucommia ulmoides via tissue culture includes the following steps:

[0009] S1: Use the current year's semi-lignified stem segments with axillary buds from superior perennial Eucommia ulmoides strains in the wild as explants;

[0010] S2: The explants from S1 were surface sterilized, cut into stem segments with axillary buds, and inoculated into axillary bud induction medium to induce axillary bud germination.

[0011] S3: Cut the axillary buds that have sprouted in S2 into segments and inoculate them into a culture medium containing plant growth regulators for the induction and proliferation of adventitious buds.

[0012] S4: The adventitious buds after proliferation culture are induced to root in a two-step method inside the bottle or outside the bottle to obtain complete regenerated Eucommia ulmoides plants with robust root systems.

[0013] Beneficial Effects: This invention uses axillary buds that have germinated for a certain period as explants. Stem segments with axillary buds of suitable size are cut and inoculated into a culture medium supplemented with plant growth regulators to induce and elongate adventitious buds. These segments are then inoculated into a culture medium supplemented with plant growth regulators for in vitro adventitious root induction or ex vitro rooting. Through transplanting and seedling management, complete regenerated Eucommia ulmoides plants are ultimately obtained, enabling the rapid propagation of superior Eucommia ulmoides strains. This method can quickly obtain high-quality Eucommia ulmoides seedlings, solving the problem of traditional seedling cultivation being limited by the availability of suitable propagation materials, and providing technical support for the sustainable development of the Eucommia ulmoides industry.

[0014] Preferably, the surface disinfection of the explant in step S2 includes the following steps: cutting the explant into 4-6cm segments, placing them in a culture bottle, sealing the bottle with gauze, rinsing under running water for 20 minutes, then wiping with 75% anhydrous ethanol once, rinsing with sterile water three times in a sterile operating table, then disinfecting with 75% anhydrous ethanol for 45 seconds, rinsing with sterile water three times, then disinfecting with 0.1% mercuric chloride solution for 3 minutes, and finally rinsing with sterile water six times; shaking continuously during disinfection, and absorbing surface moisture after disinfection.

[0015] Preferably, the axillary bud induction medium in step S2 comprises: DKW, 0.5-3.0 mg / L ZT, 30 g / L sucrose, and 6.8 g / L agar.

[0016] Preferably, in step S3, the sprouted axillary buds are cut into segments with a length of 0.8-1.2 cm.

[0017] Preferably, in step S3, the culture medium containing plant growth regulators refers to DKW culture medium containing 0.5-2.0 mg / L 6-BA, 0.5-2.0 mg / L ZT, 30 g / L sucrose, and 6.8 g / L agar.

[0018] Preferably, the two-step rooting method in bottle in step S4 refers to first pre-culturing 2.5-3.5cm adventitious buds in DKW rooting medium containing 0-5.0mg / LNAA, 20g / L sucrose and 6.8g / L agar for 0-9 days, and then transferring them to 1 / 2 DKW medium without hormones and with different concentrations of AC added to continue rooting, and then obtaining complete regenerated Eucommia ulmoides plants through transplantation and acclimatization.

[0019] Preferably, in step S4, the in vitro rooting induction refers to planting the morphological lower end of the adventitious buds in a seedling tray nutrient substrate containing 0.5-2.0 mg / L NAA and 1 / 4 DKW nutrient solution without organic matter to induce rooting, and then obtaining complete regenerated Eucommia ulmoides plants through seedling management; wherein the nutrient substrate is a mixture of nutrient soil and vermiculite, and the volume ratio of nutrient soil to vermiculite is 2:1.

[0020] Preferably, the seedling management involves covering the transplanted Eucommia ulmoides tissue culture seedlings and placing them in a greenhouse with a temperature of 23±2℃ and a relative humidity of 85-90% for seedling cultivation. Watering is done once every 5-7 days, and the plastic cover is removed after one month.

[0021] Preferably, the germination induction of axillary buds in S2, the induction and elongation of adventitious buds in S3, and the two-step rooting method in the bottle or the rooting induction culture conditions in the bottle in S4 are as follows: temperature 23±2℃, LED white light, photoperiod of 14h light / 10h darkness, and light intensity of 2000lx.

[0022] The advantages of this invention are:

[0023] (1) This invention uses the current year semi-lignified stem segments of superior perennial Eucommia ulmoides strains in the wild as explants, and achieves in vitro regeneration of Eucommia ulmoides plants through direct adventitious bud induction. It has the characteristics of convenient material acquisition, abundant materials, and the ability to maintain the superior traits of Eucommia ulmoides parent strains.

[0024] (2) The adventitious bud induction and elongation of the present invention are carried out simultaneously, which simplifies the operation and shortens the cultivation time, effectively improving the production efficiency and quality of regenerated plants.

[0025] (3) The propagation process uses stem segments to directly induce adventitious buds, which has a high regeneration efficiency and a 100% segment induction rate. After 4 weeks, the induced adventitious buds are 4.1 cm tall on average and can be reproduced into 4 segments, resulting in a high propagation coefficient. After the adventitious buds elongate, on the one hand, an in-bottle adventitious root induction method is adopted, using a two-step rooting method. Roots can be induced in 11 days, which greatly shortens the rooting time. The adventitious root induction rate can reach up to 93.02%, and there is less callus at the root base, which lays the foundation for later molecular genetic improvement. On the other hand, direct external rooting is carried out, which improves the propagation coefficient while avoiding in-bottle rooting of tissue culture seedlings and subsequent acclimatization and transplanting, greatly reducing the complexity of propagation and shortening the propagation cycle. Attached Figure Description

[0026] Figure 1 The image shows the axillary bud elongation of stem segments from superior wild Eucommia ulmoides strains in different concentrations of ZT medium in Example 1 of this invention.

[0027] Figure 2 for Figure 1A comparison of the lengths of adventitious buds corresponding to different concentrations of ZT after 4 weeks of axillary bud culture;

[0028] Figure 3 This is a diagram showing the elongation of Eucommia ulmoides tissue culture stem segments after 4 weeks of induction of adventitious buds in ZT medium of different concentrations in Example 2 of the present invention.

[0029] Figure 4 for Figure 3 A comparison of the lengths of adventitious shoots corresponding to different concentrations of ZT after 4 weeks of adventitious shoot culture;

[0030] Figure 5 This is a diagram showing the elongation of Eucommia ulmoides tissue culture stem segments after 4 weeks of induction of adventitious buds in different concentrations of 6-BA medium in Example 2 of the present invention.

[0031] Figure 6 for Figure 5 A comparison of the lengths of adventitious shoots corresponding to different concentrations of 6-BA after 4 weeks of adventitious shoot culture;

[0032] Figure 7 This is a diagram of the two-step rooting process of Eucommia ulmoides tissue culture elongation buds in Example 3 of the present invention (stereoscopic microscope);

[0033] Figure 8 These are real-life photos of the in-bottle rooting process of Eucommia ulmoides tissue culture elongation shoots in different treated culture media in Example 3 of the present invention.

[0034] Figure 9 The average number of adventitious roots induced in Eucommia ulmoides tissue culture elongation shoots after treatment with different concentrations of NAA for different number of days in Example 3 of this invention;

[0035] Figure 10 The average adventitious root length induced in Eucommia ulmoides tissue culture elongation shoots after treatment with different concentrations of NAA for different number of days in Example 3 of this invention;

[0036] Figure 11 The adventitious root induction rate of Eucommia ulmoides tissue culture elongation shoots after treatment with different concentrations of NAA for different number of days in Example 3 of the present invention;

[0037] Figure 12 This is a photograph taken two months after the Eucommia ulmoides tissue culture bottle rooted in the culture medium and then transplanted and acclimatized, according to Example 3 of the present invention.

[0038] Figure 13 This is a diagram of the external rooting of Eucommia ulmoides tissue culture elongation shoots transplanted into a mixed substrate tray in Example 4 of the present invention (1 month).

[0039] Figure 14 The complete regenerated Eucommia ulmoides plant (1 month) obtained after the Eucommia ulmoides buds were exogenously rooted in the septum of the present invention in Example 4;

[0040] Figure 15This refers to the average number of adventitious roots of Eucommia ulmoides tissue culture elongation shoots after 4 weeks of in-bottle rooting in different concentrations of culture medium in Example 5 of the present invention.

[0041] Figure 16 This is a comparison of the average adventitious root length of Eucommia ulmoides tissue culture elongation shoots after 4 weeks of in-bottle rooting in different concentrations of culture medium in Example 5 of the present invention. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0044] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0045] In the following examples, the Eucommia ulmoides seedlings used were provided by Henan Flora Agricultural Technology Co., Ltd., and the variety was Qin Zhong No. 1, which is a perennial high-quality seedling.

[0046] Example 1:

[0047] The specific steps for obtaining aseptic seedlings of superior Eucommia ulmoides strains are as follows:

[0048] (1) Material selection: such as Figure 1 The current-year semi-lignified stem segments with axillary buds of the superior perennial Eucommia ulmoides strains shown are used as explants.

[0049] (2) Surface disinfection and axillary bud germination induction:

[0050] The explants in (1) were surface disinfected, cut into stem segments with axillary buds, and inoculated into axillary bud induction medium to induce axillary bud germination.

[0051] The explants were cut into 5cm segments and placed in 300ml plastic culture bottles. After sealing with gauze, they were rinsed under running water for 20 minutes. Then, they were wiped with 75% anhydrous ethanol for disinfection, placed in a sterile operating table and rinsed three times with sterile water. They were then disinfected with 75% anhydrous ethanol for 45 seconds, rinsed three times with sterile water, disinfected with 0.1% mercuric chloride solution for 3 minutes, and finally rinsed six times with sterile water. The bottles were shaken continuously during disinfection, and the surface moisture was dried after disinfection.

[0052] Necrotic parts at both ends of the stem segment were removed, and the stem segment was then cut into 1cm segments, each with at least one axillary bud. These segments were then inoculated into axillary bud induction medium containing DKW, 0.5-3.0 ZT, 30 g / L sucrose, and 6.8 g / L agar. Germination induction culture of the axillary buds was conducted in a culture room at 23±2℃ under LED white light, with a photoperiod of 14h light / 10h dark and a light intensity of 2000 lx. After 4 weeks of culture, the axillary bud germination rate was 100%. Results are as follows: Figure 1 and 2 As shown, different concentrations of ZT significantly promoted the induction of axillary buds of Eucommia ulmoides. With the increase of ZT concentration, the adventitious buds of Eucommia ulmoides grew taller and taller, with an average maximum height of 3.48 cm.

[0053] Example 2:

[0054] The specific procedures for inducing and elongating adventitious buds of Eucommia ulmoides are as follows:

[0055] (1) Material selection: such as Figure 1 The current-year semi-lignified stem segments with axillary buds of the superior perennial Eucommia ulmoides strains shown are used as explants.

[0056] (2) Surface disinfection and axillary bud germination induction:

[0057] The explants in (1) were surface disinfected, cut into stem segments with axillary buds, and inoculated into axillary bud induction medium for axillary bud germination induction. The explants were cut into 5cm segments, placed in 300ml plastic culture bottles, sealed with gauze, and rinsed under running water for 20min. Then, they were wiped with 75% anhydrous ethanol, placed in a sterile operating table and rinsed with sterile water 3 times, then disinfected with 75% anhydrous ethanol for 45s, then rinsed with sterile water 3 times, then disinfected with 0.1% mercuric chloride solution for 3min, and finally rinsed with sterile water 6 times. During the disinfection, the explants were shaken continuously, and the surface moisture was dried after disinfection. The necrotic parts at both ends of the stem segments were removed, and the stem segments were cut into 0.8cm segments with at least one axillary bud for later use. The segments were inoculated into axillary bud induction medium, the components of which included: DKW, 2.0mg / LZT, 30g / L sucrose and 6.8g / L agar.

[0058] (3) Expand cultivation:

[0059] Using the axillary buds that germinated 4 weeks after step (2) as the source of explants, cut them into segments with a length of 1.0 cm and axillary buds, and inoculate them into a culture medium supplemented with plant growth regulators, which is DKW medium supplemented with 0.5-2.0 mg / L 6-BA or 0.5-2.0 mg / L ZT, 30 g / L sucrose and 6.8 g / L agar; the germination and induction culture of the axillary buds of the stem segments were carried out in a culture room with a temperature of 23±2℃, LED white light, a photoperiod of 14h light / 10h dark, and a light intensity of 2000 lx.

[0060] like Figure 3 and 5 As shown, different concentrations of 6-BA and ZT significantly promoted the induction and elongation of adventitious buds in Eucommia ulmoides; statistical results revealed that ( Figure 4 and 6 ZT had a stronger germination induction effect on axillary buds of Eucommia ulmoides than 6-BA. As the concentration of ZT increased, the height of adventitious buds became higher and higher. Considering the overall cost, ZT 2.0 mg / L was selected as the optimal concentration. However, the higher the concentration of 6-BA, the less significant the induction effect was. Moreover, as the concentration increased, the leaves of adventitious buds curled. The ZT group performed better than the 6-BA group.

[0061] Example 3:

[0062] The two-step rooting method for adventitious buds in tissue culture of Eucommia ulmoides is as follows:

[0063] (1) Material selection: such as Figure 1 The current-year semi-lignified stem segments with axillary buds of the superior perennial Eucommia ulmoides strains shown are used as explants.

[0064] (2) Surface disinfection and axillary bud germination induction:

[0065] The explants in (1) were surface disinfected, cut into stem segments with axillary buds, and inoculated into axillary bud induction medium for axillary bud germination induction. The explants were cut into 4cm segments, placed in 300ml plastic culture bottles, sealed with gauze, and rinsed under running water for 20min. Then, they were wiped with 75% anhydrous ethanol, placed in a sterile operating table and rinsed with sterile water 3 times, then disinfected with 75% anhydrous ethanol for 45s, then rinsed with sterile water 3 times, then disinfected with 0.1% mercuric chloride solution for 3min, and finally rinsed with sterile water 6 times. During the disinfection, the explants were shaken continuously, and the surface moisture was dried after disinfection. The necrotic parts at both ends of the stem segments were removed, and the stem segments were cut into 1.2cm segments with at least one axillary bud for later use. The segments were inoculated into axillary bud induction medium, the components of which included: DKW, 2.0mg / LZT, 30g / L sucrose and 6.8g / L agar.

[0066] (3) Expand cultivation:

[0067] Using the axillary buds that germinated 4 weeks after step (2) as the source of explants, cut them into segments with a length of 1.0 cm and axillary buds, and inoculate them into a culture room with a culture medium containing plant growth regulators, which is DKW medium containing 2.0 mg / L ZT, 30 g / L sucrose and 6.8 g / L agar; to induce the germination of axillary buds of stem segments.

[0068] (4) Two-step rooting of adventitious buds in a bottle:

[0069] The two-step in-bottle rooting method involves first pre-culturing 2.5-3.5cm adventitious buds in DKW rooting medium (0-5.0mg / L NAA, 20g / L sucrose, and 6.8g / L agar) for 0-9 days, then transferring them to hormone-free 1 / 2 DKW medium for continued rooting. After 4 weeks of cultivation in a greenhouse at 23±2℃, under LED white light with a photoperiod of 14h light / 10h dark and a light intensity of 2000lx, the induction rate of adventitious roots, the average number of adventitious roots produced per explant, and the average length of adventitious roots are calculated.

[0070] like Figure 7 As shown, under a stereomicroscope, it was observed that the base of the adventitious bud stem began to swell on day 3, subsequently continuing to swell and produce callus tissue, and adventitious roots began to appear on day 11. Figure 5 The diagram shows the rooting process of Eucommia ulmoides adventitious buds in a flask, divided into 3 groups: Group 1 (1 / 2 group) is CK, which does not undergo NAA treatment and directly roots in 1 / 2 DKW medium; Group 2 (N3-1 / 2 group) is pre-cultured in 3.0 mg / L NAA rooting medium for 7 days, and then transferred to hormone-free 1 / 2 DKW medium to continue rooting; Group 3 (N3 group) is cultured in 3.0 mg / L NAA rooting medium continuously. Figure 8 The results showed that the 1 / 2 group did not induce roots after 4 weeks of culture in 1 / 2 DKW medium, and only a little callus was produced at the base; while the N3 group could induce adventitious roots, but it took longer, the roots were shorter and the callus at the base was larger, which affected root development and subsequent transplanting. Figure 9 , 10Studies 11 and 11 showed that NAA concentration and pretreatment days had a significant impact on adventitious root induction. With increasing NAA concentration (0-5.0 mg / L) and pretreatment days (0-9 days), the average number of adventitious roots, average root length, and induction rate all showed a trend of first increasing and then decreasing. Among all experimental groups, the group pretreated with 5.0 mg / L NAA for 7 days had the highest average number of adventitious roots, reaching 6.4 roots. However, when pretreated with NAA at various concentrations for 1 day, the average number of adventitious roots in all experimental groups did not exceed 2. The longest average adventitious root length was 2.93 cm. The highest adventitious root induction rate reached 93.02%, while the lowest was less than 50%. We speculate that the effect of auxin NAA on Eucommia ulmoides rooting is only in the early stimulation stage (approximately 5-7 days), while high concentrations of NAA actually inhibit root elongation in the subsequent stages. Figure 12 The image shows a complete regenerated Eucommia ulmoides tissue culture plant that has been transplanted and acclimatized after rooting in a bottle.

[0071] Example 4:

[0072] The specific procedures for exogenous rooting of adventitious buds from Eucommia ulmoides tissue culture bottles are as follows:

[0073] (1) Material selection: such as Figure 1 The current-year semi-lignified stem segments with axillary buds of the superior perennial Eucommia ulmoides strains shown are used as explants.

[0074] (2) Surface disinfection and axillary bud germination induction:

[0075] The explants in (1) were surface-sterilized, cut into stem segments with axillary buds, and inoculated into axillary bud induction medium for axillary bud germination induction. The explants were cut into 6cm segments, placed in 300ml plastic culture bottles, sealed with gauze, and rinsed under running water for 20min. Then, they were wiped once with 75% anhydrous ethanol, placed on a sterile operating table, rinsed three times with sterile water, then sterilized with 75% anhydrous ethanol for 45s, then rinsed three times with sterile water, then sterilized with 0.1% mercuric chloride solution for 3min, and finally rinsed six times with sterile water. During the sterilization process, the explants were shaken continuously, and after sterilization, the surface moisture was dried.

[0076] Then, the necrotic parts at both ends of the stem segment were removed, and the stem segment was cut into segments of 1.2 cm in size with at least one axillary bud for later use. The segments were then inoculated into axillary bud induction medium, the components of which included: DKW, 2.0 mg / L ZT, 30 g / L sucrose and 6.8 g / L agar.

[0077] (3) Expand cultivation:

[0078] Using the axillary buds that germinated 4 weeks after step (2) as the source of explants, cut them into segments with a length of 1.0 cm and axillary buds, and inoculate them into a culture medium supplemented with plant growth regulators, which is DKW medium supplemented with 2.0 mg / L ZT, 30 g / L sucrose and 6.8 g / L agar; the germination induction culture of the stem segment axillary buds was carried out in a culture room with a temperature of 23±2℃, LED white light, a photoperiod of 14h light / 10h dark, and a light intensity of 2000 lx.

[0079] (4) Rooting of adventitious buds outside the bottle:

[0080] Once the adventitious buds from step (3) have elongated to 2.5-3.5 cm, separate them from the tissue culture bottle, trim them into terminal buds with 2-3 true leaves, and morphologically transplant their lower ends into a seedling tray substrate sprayed with 0.5-2.0 mg / L NAA and 1 / 4 DKW nutrient solution without organic matter. Cover with a plastic cap and induce adventitious roots outside the bottle. The nutrient substrate is a mixture of nutrient soil and vermiculite, with a volume ratio of 2:1. Place the trays in a greenhouse at a temperature of 23±2℃ and a relative humidity of 85-90% for seedling cultivation, watering every 5-7 days. Remove the plastic cap after one month. Results: The highest induction rate of adventitious bud rooting in vitro was 90.6%, with good growth, and seedlings were formed within one month (e.g., Figure 13 and 14 This method avoids the rooting of tissue culture seedlings in bottles and the subsequent acclimatization and transplanting, greatly reducing the complexity of propagation and shortening the propagation cycle.

[0081] Example 5:

[0082] This example tested the effect of different concentrations of activated carbon on the rooting of Eucommia ulmoides adventitious buds in flasks. The inventors only changed the concentration of activated carbon. The obtained elongated Eucommia ulmoides tissue culture adventitious buds were pre-cultured for 7 days in DKW rooting medium containing 3.0 mg / L NAA, 20 g / L sucrose, and 6.8 g / L agar. Then, they were transferred to 1 / 2 DKW medium without hormones and supplemented with different concentrations of activated carbon (0.2 g / L, 0.5 g / L, 1.0 g / L, and 2.0 g / L) to continue rooting. The remaining steps were the same as in Example 3. After culturing in a greenhouse for 4 weeks, the effect of different concentrations of activated carbon on the rooting of elongated Eucommia ulmoides buds in flasks was statistically analyzed. The results are as follows: Figure 15 and 16 As shown, the exogenous addition of AC has a significant effect on the rooting of Eucommia ulmoides buds in the culture medium. Within a certain concentration range, AC was found to inhibit the number of adventitious roots, but with the increase of AC concentration, the average root length of adventitious roots gradually increased, reaching a maximum of 3.27 cm at an AC concentration of 2.0 g / L. Furthermore, the addition of AC to the culture medium resulted in greener leaves and better growth of the regenerated Eucommia ulmoides seedlings.

[0083] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for rapid seedling cultivation of Eucommia ulmoides via tissue culture, characterized in that, Includes the following steps: S1: Use the current year's semi-lignified stem segments with axillary buds from superior perennial Eucommia ulmoides strains in the wild as explants; S2: The explants from S1 are surface-sterilized, cut into stem segments with axillary buds, and inoculated into axillary bud induction medium to induce axillary bud germination; the axillary bud induction medium consists of DKW, 0.5-3.0 mg / L ZT, 30 g / L sucrose, and 6.8 g / L agar; S3: Cut the axillary buds that have germinated in S2 into segments and inoculate them into a culture medium supplemented with plant growth regulators for the induction and proliferation of adventitious buds; the culture medium supplemented with plant growth regulators is DKW medium containing 0.5-2.0 mg / L 6-BA, 30 g / L sucrose and 6.8 g / L agar. or DKW medium containing 0.5-2.0 mg / L ZT, 30 g / L sucrose, and 6.8 g / L agar; S4: The adventitious buds after proliferation culture are induced to root in a two-step method inside the bottle or outside the bottle to obtain complete regenerated Eucommia ulmoides plants with robust root systems. The two-step rooting method in bottles refers to first pre-culturing 2.5-3.5cm adventitious buds in DKW rooting medium containing 3-5mg / LNAA, 20g / L sucrose and 6.8g / L agar for 7-9 days, then transferring them to hormone-free 1 / 2 DKW medium to continue rooting, and finally obtaining complete regenerated Eucommia ulmoides plants through transplanting and acclimatization. The aforementioned in vitro rooting induction refers to planting the morphological lower end of adventitious buds in a seedling tray nutrient substrate containing 0.5-2.0 mg / L NAA and 1 / 4 DKW nutrient solution without organic matter to induce rooting, and then obtaining complete regenerated Eucommia ulmoides plants through seedling management; wherein the nutrient substrate is a mixture of nutrient soil and vermiculite, and the volume ratio of nutrient soil to vermiculite is 2:

1.

2. The method for rapid seedling cultivation of Eucommia ulmoides via tissue culture according to claim 1, characterized in that, The surface disinfection of explants in step S2 includes the following steps: cut the explants into 4-6 cm segments, place them in a culture bottle, seal the bottle with gauze, rinse under running water for 20 minutes, then wipe them once with 75% anhydrous ethanol, rinse them three times with sterile water in a sterile operating table, disinfect them again with 75% anhydrous ethanol for 45 seconds, rinse them three times with sterile water, then disinfect them with 0.1% mercuric chloride solution for 3 minutes, and finally rinse them six times with sterile water; shake continuously during disinfection, and dry the surface moisture after disinfection.

3. The method for rapid seedling cultivation of Eucommia ulmoides via tissue culture according to claim 1, characterized in that, The axillary bud induction medium in step S2 consists of DKW, 2.0 mg / L ZT, 30 g / L sucrose, and 6.8 g / L agar.

4. The method for rapid seedling cultivation of Eucommia ulmoides via tissue culture according to claim 1, characterized in that, In step S3, the sprouted axillary buds are cut into segments with a length of 0.8-1.2cm.

5. The method for rapid seedling cultivation of Eucommia ulmoides via tissue culture according to claim 1, characterized in that, In step S3, the culture medium containing plant growth regulators is DKW medium containing 2.0 mg / L ZT, 30 g / L sucrose, and 6.8 g / L agar.

6. The method for rapid seedling cultivation of Eucommia ulmoides via tissue culture according to claim 1, characterized in that, In step S3, the culture medium containing plant growth regulators is DKW medium containing 1.0 mg / L ZT, 30 g / L sucrose, and 6.8 g / L agar.

7. The method for rapid seedling cultivation of Eucommia ulmoides via tissue culture according to claim 1, characterized in that, The two-step rooting method in bottle in step S4 refers to first pre-culturing 2.5-3.5cm adventitious buds in DKW rooting medium composed of 3.0mg / LNAA, 20g / L sucrose and 6.8g / L agar for 7 days, and then transferring them to hormone-free 1 / 2DKW medium to continue rooting. Finally, complete regenerated Eucommia ulmoides plants are obtained through transplanting and acclimatization.

8. The method for rapid seedling cultivation of Eucommia ulmoides via tissue culture according to claim 1, characterized in that, The seedling management involves covering the transplanted Eucommia ulmoides tissue culture seedlings and placing them in a greenhouse with a temperature of 23±2℃ and a relative humidity of 85-90% for seedling cultivation. Watering is done once every 5-7 days, and the plastic cover is removed after one month.

9. The method for rapid seedling cultivation of Eucommia ulmoides via tissue culture according to claim 1, characterized in that, The germination induction of axillary buds described in S2, the induction and elongation of adventitious buds described in S3, and the two-step rooting method in the bottle or the rooting induction method outside the bottle described in S4 are all under the following conditions: temperature 23±2℃, LED white light, photoperiod of 14h light / 10h darkness, and light intensity of 2000lx.

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