Establishment method and application of efficient regeneration system of inula flower leaves

Abstract

The invention discloses an establishment method and application of an efficient regeneration system of inula flower leaves. The method comprises the following steps: (1) cutting sterile inula flower leaves into small blocks of 1 cm < 2 >, and sequentially putting the small blocks into a cluster bud induction culture medium and a rooting culture medium for culturing; and (2) taking out the rooting aseptic seedlings with the height of 1-3 cm from the culture bottle, washing off a root culture medium, and transplanting the seedlings into a matrix of nutrient soil to obtain complete regenerated plants. According to the method, the inula flower leaves are selected as the explants for plant tissue regeneration culture, required materials are low in cost and easy to obtain, plant growth is not damaged, the operation process is simple and easy to implement, the propagation coefficient is high, the time period is short, and the method can be used for large-scale propagation and cultivation of inula flower seedlings. In addition, the efficient regeneration system of the inula flower leaves provides an important basic material for gene editing and inula flower germplasm innovation.

Description

Technical Field

[0001] The invention belongs to the technical field of modern Chinese herbal medicine cultivation, and particularly relates to a method for establishing and applying an efficient regeneration system of Inula japonica leaves. Background Art

[0002] Inula japonica Thunb. is a traditional Chinese medicine. The 2020 edition of the Chinese Pharmacopoeia records that Inula japonica Thunb. has the effects of lowering qi, eliminating phlegm, promoting diuresis, and stopping vomiting. Modern studies have shown that Inula japonica Thunb. has anti-tumor and anti-inflammatory activities. It is recommended as an anti-tumor Chinese herbal medicine and has been included in the medical insurance list. Therefore, Inula japonica Thunb. has important economic value in the production of Chinese herbal medicines.

[0003] Under natural conditions, Inula mainly propagates by root suckers, which has the advantages of rapid growth and the ability to maintain the excellent properties of the mother tree, but has the disadvantage of being limited in quantity and unable to meet the needs of large-scale cultivation. Tissue culture methods can obtain a large number of plants in a short period of time and have been widely used in recent years. For example, patent application CN106386512A uses the stem segments of Inula that were grown that year as explants to establish a method for rapid propagation of Inula. However, the explant propagation efficiency of this method is relatively low. At the same time, using the stem segments of Inula as explants seriously damages the Inula plant, which is not conducive to large-scale cultivation.

[0004] In view of the above problems, the present invention establishes a method for efficiently inducing clustered buds from Inula leaves and forming seedlings. Mature leaves that cause the least harm to the growth of Inula plants are used as explants to directly induce clustered buds. After the induced clustered buds grow to 1 to 3 cm, they are directly induced to take root. After taking root, they can be directly transplanted into seedlings without hardening. The present invention greatly improves the regeneration efficiency and rooting efficiency of Inula, obtains a new efficient propagation method of Inula, solves the shortcomings of the current Inula leaf regeneration system, lays a foundation for the genetic transformation and gene editing of Inula, and contributes to the molecular breeding and rapid propagation of Inula. Summary of the invention

[0005] The purpose of the present invention is to provide a method for establishing an efficient regeneration system of Inula leaf, which uses Inula leaf as explant to directly induce the production of clustered buds, has a high reproduction coefficient and a short time period, can be used for large-scale reproduction and cultivation of Inula seedlings, and provides important basic materials for genetic transformation, gene editing and germplasm innovation of Inula.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for establishing an efficient regeneration system of Inula japonica leaves comprises the following steps:

[0008] A. Inoculating sterile Inula japonica leaves as explants onto a cluster bud induction medium for induction culture to obtain cluster buds; the cluster bud induction medium is composed of MS medium + 0.5-2 mg / L 6-BA + 0.5-2 mg / L IBA + sucrose + plant gel;

[0009] B. Cutting the clustered buds obtained in A and transferring them to a rooting medium for rooting culture to obtain rooted seedlings; the rooting medium is composed of MS medium + 0-1 mg / L NAA + sucrose + plant gel;

[0010] C. Transplant and cultivate the rooted sterile seedlings 1 to 3 cm in height.

[0011] Preferably, the explants are obtained by cutting sterilized Inula japonica seedling leaves along the main vein direction, with each leaf being cut into 2 to 4 explants.

[0012] Preferably, the cluster bud induction medium is composed of MS medium + 1 mg / L 6-BA + 0.5 mg / L IBA + 30 g / L sucrose + 4 g / L plant gel.

[0013] Preferably, the rooting medium composition is MS medium + 0.5 mg / L NAA + 30 g / L sucrose + 4 g / L plant gel.

[0014] Preferably, the conditions for the cluster bud induction culture or rooting culture are 2000 lx of light, 16 / 8h light / dark conditions, and a temperature of 26°C.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] Compared with the existing rapid propagation methods of Inula japonica, this method has the following three advantages: first, compared with the regeneration system induced by stem segments and axillary buds, the regeneration system induced by leaves can differentiate more cluster buds, thereby improving the acquisition rate of cluster buds, that is, one explant leaf can induce 37 cluster buds on average, and the number of induced cluster buds is 6 times higher than that reported previously (Bian Yongmei, Li Sen, Liu Mengying, et al. Research on tissue culture and asexual system establishment of Inula japonica [J]. Agriculture and Technology, 2008, (01): 36-40.); second, this method greatly saves time and cost, that is, it eliminates the tedious steps of first inducing callus tissue and then differentiating callus into cluster buds; third, the cluster buds induced by this method can be directly transplanted into nutrient soil after taking root, thus eliminating the process of cluster bud seedling strengthening, which also saves time and cost. Finally, a large number of Inula japonica can be successfully obtained in 2 months. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the screening result of the Inula japonica leaf cluster bud induction culture medium in Example 1.

[0018] Figure 2 This is the optimization result of the Inula japonica leaf cluster bud induction culture medium in Example 1.

[0019] Figure 3 This is the screening result of the Inula japonica bud rooting culture medium in Example 2.

[0020] Figure 4 It is a complete flow chart of the regeneration of Inula leaf into seedlings in Example 3. DETAILED DESCRIPTION

[0021] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0022] The hormones in the present invention are referred to as follows:

[0023] 6-Benzylaminoadenine: 6-BA;

[0024] Indolebutyric acid: IBA;

[0025] Naphthaleneacetic acid: NAA;

[0026] Zeatin: ZT;

[0027] Dichlorophenylacetic acid: 2,4-D.

[0028] The present invention is further described in detail below in conjunction with specific embodiments.

[0029] The source of the sterile Inula japonica involved in the following embodiments: the sterile Inula japonica leaves used are derived from sterilized Inula japonica leaves, which are collected from the Medicinal Botanical Garden of Hubei University of Chinese Medicine, and the leaves are collected in May 2023.

[0030] Example 1: Screening and optimization of cluster shoot induction medium

[0031] In order to explore the induction effect of different hormone combinations on the clustered buds of Inula japonica leaves, two cytokinins 6-BA (0.5-2 mg / L) and ZT (0.5-2 mg / L) were used with three auxins IBA (0.5-2 mg / L), NAA (0.5-1 mg / L), and 2,4-D (1-5 mg / L) to form 12 combinations (specific hormone concentrations are shown in Table 1 below), named Y-1 to Y-12, respectively. The specific implementation plan is as follows: sterile Inula japonica leaves were cut horizontally along the main veins, and each leaf was cut into 3 small leaflets, each of which was about 1 cm in size. 2Then the treated leaves were inoculated into cluster bud induction medium (MS, 12 hormone combinations, 30g / L sucrose, 4g / L plant gel) to induce differentiation (light intensity 2000lx, 16 / 8h light / dark conditions, temperature 26℃) and the results were observed after 3 weeks. The results showed that the combination of 6-BA and IBA hormones promoted the differentiation of cluster buds ( Figure 1 a); 6-BA and NAA hormone combination promoted a small amount of differentiation of clustered buds ( Figure 1 b); 6-BA and 2,4-D hormone combination mainly promotes callus formation ( Figure 1 c); ZT and IBA hormone combination promoted rooting ( Figure 1 d); ZT and NAA hormone combination mainly promotes callus formation ( Figure 1 e); ZT and 2,4-D hormone combination mainly promotes callus formation ( Figure 1 f). Therefore, the combination of hormone 6-BA and IBA has the best effect on the induction of clustered buds of Inula japonica.

[0032] Table 1 Hormone concentrations and induction results in the bud induction medium

[0033]

[0034]

[0035] In order to improve the induction efficiency of clustered buds, we further optimized the concentrations of 6-BA and IBA (named YH-1 to YH-6, respectively). The specific optimized hormone concentrations are shown in Table 2. The specific experimental operations were consistent with the above experimental operations. The results showed that the average number of buds in the YH-1 group was 16, the average number of buds in the YH-2 group was 29, the average number of buds in the YH-3 group was 26, the average number of buds in the YH-4 group was 26, the average number of buds in the YH-5 group was 28, and the average number of buds in the YH-6 group was 37 (see Appendix for the results). Figure 2 ). Therefore, the YH-6 group had the best induction effect on the clustered buds of Inula japonica.

[0036] Table 2 Hormone concentrations and induction results in the optimized culture medium for clustered buds

[0037]

[0038] In summary, YH-6 is the optimal hormone combination for cluster bud differentiation of Inula japonica. The average number of cluster buds differentiated from one Inula japonica leaf is 37.

[0039] Example 2: Screening of Rooting Culture Medium

[0040] In order to explore the rooting medium of clustered buds, different concentrations of NAA (0-1 mg / L) were selected and named SG-1 to SG-3. The specific hormone concentrations of each group are shown in Table 3. The experimental method is to cut the clustered buds obtained in Example 1 and transfer them into the rooting medium (MS, SG-1-SG-3, 30 g / L sucrose, 4 g / L plant gel, and the preparation method is to add sucrose and plant gel to the MS medium and then sterilize it, and add the filtered and sterilized NAA solution when the temperature drops to about 50°C) for rooting culture (light intensity 2000lx, 16 / 8h light / dark conditions, temperature 26°C), and observe after 2 weeks. The results showed that the average number of roots in the SG-1 group was 4, the average number of roots in the SG-2 group was 6, and the average number of roots in the SG-3 group was 3 (see Appendix). Figure 3 ). Therefore, SG-2 is the optimal hormone concentration screened to promote the rooting of cluster buds. The average number of roots of a cluster bud of Inula japonica is 6, and the rooting rate reaches 100%.

[0041] Table 3 Hormone concentrations and rooting results in clustered shoot rooting medium

[0042]

[0043] Example 3: Transplantation of Inula japonica regenerated seedlings

[0044] In order to quickly obtain regenerated seedlings of Inula japonica, we omitted the process of strengthening and hardening the tissue culture seedlings, and directly took out the tissue culture seedlings (about 2 to 3 cm in height) that had undergone clustered bud rooting culture for 2 weeks from the culture bottle, washed off the root culture medium, and transplanted them into a matrix mixed with vermiculite and nutrient soil. The transplanting matrix was a mixture of vermiculite and nutrient soil in a volume ratio of 1:1. After 2 weeks of observation, the results showed that the survival rate of the Inula japonica seedlings planted in the mixed matrix of vermiculite and nutrient soil reached 100%, and they grew well, reaching a height of about 20 cm, which was about 6.7 to 10 times taller than the initial seedlings. The specific effects are shown as follows: Figure 4 .

Claims

1. A method for establishing an efficient regeneration system of Inula japonica leaves, characterized in that: The following steps are involved: A. Inoculating sterile Inula japonica leaves as explants onto a cluster bud induction medium for induction culture to obtain cluster buds; the cluster bud induction medium is composed of MS medium + 0.5-2 mg / L 6-BA + 0.5-2 mg / L IBA + sucrose + plant gel; B. Cutting the clustered buds obtained in A and transferring them to a rooting medium for rooting culture to obtain rooted seedlings; the rooting medium is composed of MS medium + 0-1 mg / L NAA + sucrose + plant gel; C. Transplant and culture the rooted sterile seedlings 1 to 3 cm in height.

2. The method according to claim 1, characterized in that The explants are obtained by cutting sterilized Inula japonica seedling leaves along the main vein direction, and each leaf is cut into 2 to 4 explants.

3. The method according to claim 1, characterized in that The cluster bud induction culture medium is composed of MS culture medium+1 mg / L 6-BA+0.5 mg / L IBA+30 g / L sucrose+4 g / L plant gel.

4. The method according to claim 1, characterized in that The rooting culture medium is composed of MS culture medium+0.5 mg / L NAA+30 g / L sucrose+4 g / L plant gel.

5. The method according to any one of claims 1 to 4, characterized in that The conditions for the cluster bud induction culture or rooting culture are 2000 lx of light, 16 / 8 h light / dark conditions, and a temperature of 26°C.

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