Method and culture medium for efficiently inducing adventitious buds of lonicera macranthoides tissue culture seedlings to regenerate plants

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method and a culture medium for efficiently inducing adventitious buds to regenerate plants from lonicera macranthoides tissue culture plants.The high-differentiation induction culture medium comprises an MS culture medium, a 1 / 2 MS culture medium, 6-BA, KT, NAA, GA, IAA, IBA and activated carbon. According to the method disclosed by the invention, leaves, stems and roots of aseptic seedlings of tissue culture seedlings of Zunyi genuine lonicera confusa (lonicera macranthoides) are taken as explants, and by changing an inoculation placement mode, less calluses are formed through control, the induction rate of adventitious buds is improved, tedious steps of intermediate over-culture are reduced, a culture medium is optimized, the variety of the culture medium is reduced, and the labor and material cost is reduced. Meanwhile, the problems of high pollution rate, high browning rate and the like in the explant disinfection process and the culture process are avoided, and industrial seedling breeding is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a method and culture medium for efficiently inducing adventitious buds to regenerate plants from tissue culture seedlings of Lonicera japonica. Background Art

[0002] Lonicera macranthoides Hand.-Mazz is a perennial semi-evergreen twining woody vine of the Caprifoliaceae family. It is widely distributed in Zunyi and has a long history of cultivation. The whole plant of Lonicera macranthoides can be used as medicine. It was officially included in the Pharmacopoeia in 2004 and is one of the original plants of Lonicera macranthoides. The authentic Lonicera macranthoides in Zunyi is a wild variety in Zunyi. It has a long flowering period and the corolla can crack. The cracked corolla is mainly used as the raw material for medicinal purposes. The Lonicera macranthoides planted in Zunyi is mainly propagated by seeds and branch cuttings. The seeds of Lonicera macranthoides are difficult to collect and have a long vegetative growth period. The vegetative growth period of cutting propagation is short, but the survival rate is low, and it is easy to show premature aging after full flowering. Seed collection and cuttings will reduce the yield of flower buds, resulting in farmers' unwillingness to keep seeds for breeding. The tissue culture of Honeysuckle mainly uses stems as explants. A few researchers use leaves or petioles as explants to obtain callus tissue and induce adventitious buds.

[0003] Some scholars have studied the tissue culture technology of Lonicera japonica. In 2013, Ye Litao et al. used the stems of Lonicera japonica as explants for culture and obtained regenerated buds. In the same year, Wang Huili et al. obtained regenerated plants of Lonicera japonica through hormone combination screening. Shu Qingli et al. disclosed a method for tissue culture of Lonicera japonica, and disclosed the modification and culture method of the basic culture medium MS, which included transition culture 1 and 2.

[0004] It can be found from the research of many scholars that their research has the same characteristics: complicated cultivation steps, long cultivation cycle and high cost.

[0005] In order to meet the production needs, a set of plant tissue culture technology that can obtain a large number of regenerated plants in a short period of time, which is not affected by the external environment and has strong controllability, is currently an urgent problem to be solved. Based on this, in 2024, Zhao Yingying et al. disclosed a rapid seedling method and culture medium for Lonicera japonica. Although this method simplifies the culture steps, the induction rate of adventitious buds is low, and the adventitious buds are thin and weak, which is not conducive to mass production and rooting induction in the later stage, and indirectly increases the cost.

[0006] Therefore, it is necessary to provide a method and culture medium for efficiently inducing adventitious buds and regenerating plants from tissue culture seedlings of Lonicera japonica, which can improve the induction coefficient of adventitious buds, cultivate strong seedlings, shorten the seedling raising period, be easy to operate, use fewer types of culture medium, and improve the rooting rate. Summary of the invention

[0007] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a method and culture medium for efficiently inducing adventitious buds and regenerating plants from tissue culture seedlings of Honeysuckle.

[0008] The invention discloses a method for efficiently inducing adventitious buds and regenerating plants from tissue culture seedlings of Honeysuckle, comprising the following steps:

[0009] Step 1: Using the leaves, stems and roots of the sterile seedlings of Lonicera japonica as explants, inoculating them in an adventitious bud induction medium in a certain inoculation method to induce loose cells, and culturing them for 28 to 40 days to differentiate and form a large number of adventitious buds;

[0010] Step 2: The formed adventitious buds are transferred to the cluster bud induction medium and cultured for 20 days to obtain cluster buds;

[0011] Step 3: culturing the obtained sprouts in a seedling medium for 14 days to form strong seedlings;

[0012] Step 4: Transfer the obtained seedlings to a rooting medium to induce root growth, which takes 28 to 40 days to form a complete plant.

[0013] This program greatly improves the rate of inducing adventitious buds, shortens the seedling cycle, simplifies the culture steps, reduces the types of hormones, makes the operation simpler and the cost lower, and improves the differentiation rate and survival rate, thereby greatly improving the practicality.

[0014] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0015] This scheme first improves on the existing technology and selects induced sterile seedling leaves, stem segments and roots as initial explants.

[0016] The adventitious bud induction medium, cluster bud induction medium, seedling medium and rooting medium constitute the high differentiation induction medium;

[0017] A high differentiation induction medium, comprising a basal medium and an additional component added to the basal medium;

[0018] Wherein, the basic culture medium is: one of MS and 1 / 2MS culture medium;

[0019] The added ingredients are: two or more of 6-BA, KT, NAA, GA, IAA, IBA and activated carbon;

[0020] During preparation, 6-BA is added to the basal culture medium to a concentration of 0.5-3.0 mg / L, KT is added to the basal culture medium to a concentration of 1.5-3.0 mg / L, IAA is added to the basal culture medium to a concentration of 0.1-0.8 mg / Lmg / L; NAA is added to the basal culture medium to a concentration of 0.5-1.5 mg / L, GA is added to the basal culture medium to a concentration of 0.2-0.4 mg / L, IBA is added to the basal culture medium to a concentration of 0.1-0.6 mg / L, and activated carbon is added to the basal culture medium to a concentration of 0.2-1.0 g / L.

[0021] Preferably, in step 1, the method for obtaining the tissue culture seedlings of Lonicera japonica comprises:

[0022] According to Zhao Yingying's method, the stem segments 2 to 3 nodes below the strong terminal bud of the current year were taken as the starting explants. After disinfection and sterilization and in vitro germination, sterile seedlings of Sterile Honeysuckle were obtained. After inducing strong adventitious roots through culture, complete sterile plants were obtained.

[0023] Preferably, the gray felt honeysuckle is a Zunyi local planting variety, that is, Zunyi authentic honeysuckle gray felt honeysuckle.

[0024] Preferably, in step 1, in the callus induction, the placement of leaves, stem segments and roots is as follows: when the stem segments are connected, the tips of the opposite leaves are removed and inserted into the culture medium or placed horizontally in the culture medium; the stem segments are cut to 2 to 5 cm and placed horizontally in the culture medium; the root treatment is the same as the stem segments; the induction amount of callus during the induction process is extremely low, and then adventitious buds are induced, the lighting time is 16 h / d, and the light intensity is 28000 Lux.

[0025] Preferably, in step 1, the adventitious bud induction medium adopts MS medium as the basal medium, and in the adventitious bud induction medium, the concentration of 6-BA is 0.5-3.0 mg / L, the concentration of KT is 1.5-3.0 mg / L, and the concentration of IAA is 0.1-0.8 mg / Lmg / L.

[0026] Preferably, in step 2, the bud cluster induction medium adopts MS medium as the basal medium, and in the bud cluster induction medium, the concentration of 6-BA is 0.2-0.8 mg / L, the concentration of IAA is 0.1-0.6 mg / LL, and the concentration of GA is 0.2-0.4 mg / L.

[0027] Preferably, in step 3, the seedling-strengthening culture medium adopts MS culture medium as the basal culture medium, and in the seedling-strengthening culture medium, the concentration of 6-BA is 0.2-0.8 mg / L, and the concentration of IBA is 0.2-0.6 mg / L.

[0028] Preferably, in step 4, the rooting medium adopts 1 / 2MS medium as the basic medium, and in the rooting medium, the concentration of 6-BA is 0.5-3.0 mg / L, the concentration of NAA is 0.2-0.6 mg / L, and the concentration of activated carbon is 0.2 g / L-0.6 g / L.

[0029] Preferably, in steps 1 to 3, the culture temperature is 22 to 25° C., the light intensity is 2500 to 2800 Lux, and the illumination time is 16 h / d;

[0030] In step 4, the culture temperature is 22-25° C., the culture is carried out in the dark for 7 days, and then the culture is switched to light culture with a light intensity of 2000-2500 Lux and a light time of 16 h / d.

[0031] The beneficial effects achieved by the present invention using the above structure are as follows:

[0032] This scheme uses the leaves, stem segments and roots of the sterile tissue culture seedlings of Zunyi authentic honeysuckle (gray felt honeysuckle) as explants. By changing the inoculation placement method, it controls the formation of less callus, improves the induction rate of adventitious buds, reduces the tedious steps of excessive cultivation in the middle, optimizes the culture medium, reduces the types of culture medium used, and reduces labor and material costs. At the same time, it avoids the explant disinfection process, the high contamination rate and browning rate during the culture process, and facilitates the breeding of factory seedlings;

[0033] In the prior art, the gray felt honeysuckle uses the stem segments with axillary buds of the current year as explants, induces budding and callus on a culture medium, and then cultures the callus for proliferation. The buds are induced or directly take root. Under this condition, the bud proliferation coefficient is low, and the buds are prone to browning during the callus induction process. It is difficult to induce adventitious buds from most calli, which prolongs the culture period and increases the difficulty of induction. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0035] Figure 1-8 This is a real shot of the rapid and highly induced adventitious bud cultivation of Honeysuckle in this scheme, including:

[0036] Figure 1 -A Original explant leaves; Figure 1 -Adventitious buds differentiated from different parts of leaf explants of B, C, and D;

[0037] Figure 2 -A original explant root; Figure 2 -B, C, and D are adventitious buds differentiated from the roots of root explants;

[0038] Figure 3-A Original explant stem segment without axillary buds; Figure 3 -B and C are adventitious buds differentiated from the original explant stem segments;

[0039] Figure 4 -A means encountering an organization; Figure 4 -B, C, and D are adventitious buds differentiated from callus tissue;

[0040] Figure 5 -A is the stem segment with axillary buds of the original explant; Figure 5 -B, C, and D are adventitious buds differentiated from the stem segments with axillary buds of the original explants;

[0041] Figure 6 -A is the adventitious buds transferred from adventitious buds to cluster buds; Figure 6 -B, C, and D are clustered buds induced by adventitious buds;

[0042] Figure 7 -A is the clustered buds transferred from clustered buds to strong buds; Figure 7 -B, C, and D are strong buds induced by clustered buds;

[0043] Figure 8 -A can be used to induce strong rooting shoots; Figure 8 -B Cut off the rooted single shoot; Figure 8 -C, D, and E are roots induced from single buds;

[0044] Fig. 9 This is the process flow chart of this scheme. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0046] A method for efficiently inducing adventitious buds and regenerating plants from tissue culture seedlings of Honeysuckle, comprising the following steps:

[0047] (1) Cut the leaves, stem segments, roots and top buds of the aseptic seedlings of Honeysuckle into pieces of 2 to 5 cm as the original explants.

[0048] (2) Inoculate the original explants on adventitious bud induction medium, such as Figures 1 to 5 (A)

[0049] (3) Cultivate adventitious buds from the original explants inoculated on the adventitious bud culture medium, such as Figures 1 to 5 (B)

[0050] (4) The induced adventitious buds are transferred to the bud induction medium to induce the buds to grow. Figure 6 As shown;

[0051] (5) Transfer the sprouts to the seedling medium and cultivate the seedlings. Figure 7 As shown;

[0052] (6) Cut the clustered buds into single buds, transfer them to rooting medium, induce roots, and cultivate them into complete plants. Figure 8 shown.

[0053] The adventitious bud induction medium uses MS medium (MS medium is the existing medium) as the basic medium, adds 6-BA to a concentration of 0.5-3.0 mg / L, KT to a concentration of 1.5-3.0 mg / L, and IAA to a concentration of 0.1-0.8 mg / Lmg / L;

[0054] The bud cluster induction medium uses MS medium as the basic medium, adds 6-BA to a concentration of 0.2-0.8 mg / L, adds IAA to a concentration of 0.1-0.6 mg / L, and adds GA to a concentration of 0.2-0.4 mg / L;

[0055] The seedling medium is a MS medium as a basic medium, 6-BA is added to a concentration of 0.2-0.8 mg / L, and IBA is added to a concentration of 0.2-0.6 mg / L;

[0056] The rooting medium is a 1 / 2MS medium (the 1 / 2MS medium is the existing medium) as a basic medium, 6-BA is added to a concentration of 0.5 to 3.0 mg / L, NAA is added to a concentration of 0.2 to 0.6 mg / L, and activated carbon is added to a concentration of 0.2 g / L to 0.6 g / L;

[0057] The specific components of MS medium and 1 / 2MS medium are shown in Table 1:

[0058] Table 1 Basic culture medium components

[0059]

[0060]

[0061] Specific operations such as Figures 1 to 5As shown, the aseptic plants of Gray Felt Honeysuckle are used as explants, and the apical buds, lateral buds, stem segments, leaves, and roots are cut into 2-5 cm pieces respectively. The use of such aseptic seedlings can save the need for explant disinfection, avoid contamination, make full use of the organs of the entire plant, and improve the utilization rate of aseptic seedlings. This method can not only save the need for explant disinfection, but also avoid the situation where taking explants alone will cause a large area of ​​Gray Felt Honeysuckle to lose its apical dominance, thereby affecting the yield of Gray Felt Honeysuckle in the base. It is not subject to time limits, and the original materials can be obtained at any time. Based on this, in order to improve the emergence rate of Gray Felt Honeysuckle and make full use of the original explants of Gray Felt Honeysuckle, tissue rapid propagation technology was introduced.

[0062] After fully consulting the information and literature, the prior art technology of tissue culture seedlings of Honeysuckle is mostly based on MS and B5 culture media. After adding different auxins and cytokinins, adventitious buds are induced, callus and buds are induced in the adventitious buds, and then the buds are taken to induce roots or the buds are reused to induce callus and buds, and then the callus is used to induce adventitious buds in a cycle. For example, Zhao Yingying. A method for rapid seedling formation and culture medium of Honeysuckle [P]. Guizhou Province: CN116267621B, 2024-04-16. Based on the content disclosed in the article, the researchers repeatedly verified the culture method of Honeysuckle and carried out tissue culture seedlings of Honeysuckle. The results showed that although Honeysuckle can induce certain adventitious buds in B5 culture medium as the basic culture medium, after adding 3-5 mg / L 6-BA, 0.5 mg / L NAA and 0.05% activated carbon, the callus is easy to brown and the bud induction rate is low.

[0063] Therefore, the researchers made improvements to the technical scheme for tissue culture of honeysuckle seedlings. Figures 1 to 5 The operation method shown in (A) is improved, wherein the whole sterile seedlings or the induced adventitious buds and roots are cut into 2-5 cm pieces, inoculated on the adventitious bud induction medium to form a small amount of callus, which then differentiates into adventitious buds;

[0064] The differentiated adventitious buds are transferred to a bud induction medium for cultivation to form cluster buds;

[0065] The cluster buds cultured in the cluster bud induction medium are divided into 1 to 3 buds, and transferred to the seedling medium for bud cultivation to obtain healthy and strong buds;

[0066] Then the clustered buds are separated into single buds, transferred to rooting medium, and cultured into complete plants;

[0067] It has been achieved that not only can the gray felt honeysuckle seedlings be obtained through transfer, but the initial explants can also be obtained unlimitedly without being restricted by explants and time. The cultivation coefficient has also been greatly increased, and the target variety seedlings can be obtained in a short time, greatly reducing the cost of seedling cultivation.

[0068] In the process of culturing sterile seedlings from explants in this scheme, the specific methods refer to the existing technical means, for example: refer to the means disclosed in the invention "A method for rapid seedling formation and culture medium of honeysuckle" published by Zhao Yingying et al. in 2023.

[0069] Experimental research:

[0070] 1. Experimental Materials

[0071] The sterile seedlings, buds and roots of Lonicera japonica Lonicera (Honeysuckle) in Zunyi area were selected as the original materials;

[0072] The basal culture medium is selected from MS and 1 / 2MS culture medium;

[0073] The added ingredients are two or more of 6-BA, NAA, IAA, IBA, KT and GA;

[0074] Preparation of culture medium

[0075] Adventitious bud induction medium: Add 6-BA, KT and IAA to MS medium;

[0076] Cluster bud induction medium: Add 6-BA, IAA and GA to MS medium;

[0077] Seedling medium: Add 6-BA and IBA to MS medium;

[0078] Rooting medium: Add 6-BA, NAA and activated carbon to 1 / 2MS medium;

[0079] 2. Experimental methods

[0080] Sterile plants, adventitious buds and roots are used as original explants, cut into 2-5 cm pieces and placed in an adventitious bud induction medium to induce a small amount of callus and a large number of adventitious buds; the tissues after the adventitious buds are induced are cut into small pieces and placed in a cluster bud induction medium to induce cluster buds; the cluster buds are cut into 1-3 plants, placed in a seedling medium to induce strong adventitious buds; the strong adventitious buds are cut into single buds, placed in a rooting medium, and roots are induced to form complete plants; the complete plants or the tissues of adventitious buds that have not been induced to have roots or only roots without adventitious buds are cut, and the above steps are repeated.

[0081] 3. Experimental content and results analysis

[0082] Study on adventitious bud induction

[0083] MS medium was used as the basic medium, and the components were added as shown in Table 2 below. Five pieces were inoculated for each treatment.

[0084] Table 2 Effect of adventitious bud induction medium

[0085]

[0086]

[0087] As shown in Table 2, numbers 4, 5, 6, 7, 9, 10, 11, 13, 14 and 15 can directly induce adventitious buds, and a small amount of callus tissue can be induced at the site of contact with the culture medium; numbers 4, 5, 11, 13, 14 and 15 have low induction differentiation coefficients, and can only induce buds at the side nodes of the stem segments with buds, and it is difficult to induce adventitious buds in leaves, stem segments and roots without nodes; numbers 6, 7, 9 and 10 can induce a small amount of loose callus in leaves, roots and stem segments, and then induce a large number of adventitious buds, and the induction coefficients are all above 40 times, among which the culture medium of number 6 can reach more than 50 times.

[0088] Table 3 Effect of bud induction medium

[0089]

[0090] Six optimized treatments were selected for the cluster bud culture medium. As can be seen from Table 3, each treatment can induce cluster buds. In treatments 1 and 2, the bud growth time is longer and the number of buds is smaller. The undifferentiated adventitious buds will be highly dispersed again during the induction process to form callus, making it difficult to form adventitious buds. Treatment 3 has a short bud formation time and a large number of buds. Treatments 4, 5 and 6 have a longer induction time, which can induce robust buds in adventitious buds to form cluster buds, and some leaves turn yellow. It can be seen that treatment 3 is the best culture medium for cluster bud induction.

[0091] Table 4 Effect of seedling medium

[0092]

[0093] Due to the large amount of adventitious buds, the induced buds are relatively weak and the induced rooting rate is low, so a step of seedling culture is added. The seedling culture medium is based on MS medium and a variety of cytokinins and auxins are added.

[0094] It can be seen from Table 4 that the type of auxin has an obvious effect on the growth of strong buds. It can be seen from culture media 1 to 3 that the strong buds cultured in culture media 2 are thick and have short internodes, which is the best material for inducing rooting. Cytokinins also have a prominent effect on buds. It can be seen from culture media 3 to 8 that 6-BA is the best cytokinin. It can be seen that based on MS culture medium, adding 6-BA and IBA can induce strong single buds, which provides excellent material for the induction of roots in the later stage.

[0095] Table 5 Effect of rooting medium

[0096]

[0097] Separate the robust clustered buds or single buds with 4 to 6 buds into single buds and transfer them to the rooting medium to induce rooting;

[0098] As shown in Table 5, when 1 / 2MS medium was used as the base medium, no cytokinin was added, and only auxin was added, adding only NAA could induce a large number of strong roots in a short time, but a large mass of callus tissue would appear at the bottom in the later stage, and the upper buds would slowly turn yellow and wilt, making it difficult to form transplantable seedlings. Adding only IAA could induce adventitious roots, and the roots stretched out from the cortex, but the rooting rate was low, the roots were slender, the buds were normal, and the growth was slow.

[0099] Adding appropriate amounts of mitogens and activated carbon can induce adventitious roots. When the activated carbon is at 0.4g / L-1.0g / L, adventitious roots can be induced in 45 days. At 0.4g / L, the roots are relatively strong and the seedlings are easy to survive.

[0100] It should be noted that, in this article, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0101] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A high differentiation induction medium, characterized in that The culture medium includes the following components: MS culture medium, 1 / 2MS culture medium, 6-BA, KT, NAA, GA, IAA, IBA and activated carbon.

2. A high differentiation induction medium according to claim 1, characterized in that: The high differentiation induction medium consists of an adventitious bud induction medium, a cluster bud induction medium, a seedling growth medium and a rooting medium; Wherein, the adventitious bud induction medium comprises the following components: MS medium, 6-BA, KT, IAA; The cluster bud induction medium comprises the following components: MS medium, 6-BA, IAA, and GA; The seedling-strengthening culture medium comprises the following components: MS culture medium, 6-BA, and IBA; The rooting medium comprises the following components: 1 / 2MS medium, 6-BA, NAA and activated carbon.

3. A high differentiation induction medium according to claim 1, characterized in that: The adventitious bud induction medium adopts MS medium as the basic medium, and 6-BA, KT and IAA are added into the basic medium to form the adventitious bud induction medium; Among them, the concentration of 6-BA in the adventitious bud induction medium is 0.5-3.0 mg / L; The concentration of KT in the adventitious bud induction medium is 1.5-3.0 mg / L; The concentration of IAA in the adventitious bud induction medium is 0.1-0.8 mg / L.

4. A high differentiation induction medium according to claim 1, characterized in that: The bud cluster induction medium adopts MS medium as the basic medium, and 6-BA, IAA and GA are added into the basic medium to form the bud cluster induction medium; Among them, the concentration of 6-BA in the bud induction medium is 0.2-0.8 mg / L; The concentration of IAA in the bud induction medium is 0.1-0.6 mg / L; The concentration of GA in the bud induction medium is 0.2-0.4 mg / L.

5. A high differentiation induction medium according to claim 1, characterized in that: The seedling-strengthening culture medium adopts MS culture medium as the basic culture medium, and 6-BA and IBA are added into the basic culture medium to form the seedling-strengthening culture medium; Among them, the concentration of 6-BA in the seedling medium is 0.2-0.8 mg / L; The concentration of IBA in the seedling culture medium is 0.2-0.6 mg / L.

6. A high differentiation induction medium according to claim 1, characterized in that: The rooting medium adopts 1 / 2MS medium as the basic medium, and 6-BA, NAA and activated carbon are added into the basic medium to form the rooting medium; Among them, the concentration of 6-BA in the rooting medium is 0.5-3.0 mg / L; The concentration of NAA in the rooting medium is 0.2-0.6 mg / L; The concentration of activated carbon in the rooting medium is 0.2g / L~0.6g / L.

7. A method for efficiently inducing adventitious bud regeneration of plants from tissue culture seedlings of Honeysuckle of Claims 1 to 6, characterized in that: The steps include: Step 1: using sterile leaves, roots and stem segments of tissue culture seedlings of Lonicera japonica as explants, inducing dispersed cells through light culture in an adventitious bud induction medium, and differentiating to form adventitious buds; Step 2: The obtained adventitious buds are transferred to a cluster bud induction medium for further cultivation to form cluster buds; Step 3: transferring the obtained clustered buds to a seedling medium for seedling growth to form seedlings; Step 4: Transfer the obtained seedlings to rooting medium to induce root system to obtain complete plants.

8. The method for efficiently inducing adventitious bud regeneration of Lonicera japonica tissue culture seedlings according to claim 7, characterized in that: In step 1, the method for obtaining the tissue culture seedlings of Lonicera japonica comprises: Cut the top 1 to 3 nodes of the healthy young shoots of the marked plants of the gray felt honeysuckle, culture them with the nodes, and obtain sterile gray felt honeysuckle tissue culture seedlings after disinfection and sterilization, and germination of new shoots in vitro. The tissue culture seedlings obtain root systems after rooting induction.

9. The method for efficiently inducing adventitious bud regeneration of Lonicera japonica tissue culture seedlings according to claim 7, characterized in that: The gray felt honeysuckle comprises sterile seedlings and leaves, stem segments with buds, stems and roots of gray felt honeysuckle.

10. The method for efficiently inducing adventitious bud regeneration of Lonicera japonica tissue culture seedlings according to claim 7, characterized in that: In step 1, the light culture is carried out at a temperature of 20-25° C., a light intensity of 2800-3000 Lux, and a light time of 16 h / d; In steps 2 and 3, the culture temperature of the bud induction medium and the seedling medium is 22 to 25°C, the light intensity is 2500 to 2800 Lux, and the light duration is 16 h / d; the culture temperature of the rooting medium is 22 to 25°C, the dark culture is performed for 7 days, and then the culture is switched to light culture, with a light intensity of 2000 to 2500 Lux and a light duration of 16 h / d.

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