Medium and method for promoting the elongation of adventitious shoots of plumeria acutifolia and tissue culture seedling culture method

Abstract

The application relates to the technical field of plant breeding, in particular to a culture medium for promoting the elongation of adventitious buds of thousand-mile fragrant plants, a method and a tissue culture seedling culture method. The culture medium for promoting the elongation of adventitious buds of thousand-mile fragrant plants comprises a bud induction culture medium, a proliferation culture medium and an elongation culture medium. The proliferation culture medium is prepared by adding 6-BA and IAA into a basic culture medium. The elongation culture medium is prepared by adding 6-BA and IAA into the basic culture medium, or adding 6-BA, GA3 and NAA into the basic culture medium, or adding 6-BA and GA3 into the basic culture medium. Thousand-mile fragrant plant explants are proliferated to obtain the adventitious buds of the thousand-mile fragrant plants, and then the elongation culture medium is used to culture the adventitious buds to obtain the elongated adventitious buds of the thousand-mile fragrant plants. The application can effectively promote the growth of the adventitious buds of the thousand-mile fragrant plants, solve the problem of slow growth of the adventitious buds of the thousand-mile fragrant plants, improve the effective bud rate and effectively shorten the culture period, so that the rooting culture of the thousand-mile fragrant plant tissue culture seedlings can be better carried out.

Description

Technical Field

[0001] This application relates to the field of medicinal plant propagation technology, specifically, to a culture medium, method, and method for promoting the elongation of adventitious buds of Murraya paniculata. Background Technology

[0002] Murraya paniculata, belonging to the Rutaceae family and the Murraya genus, is one of the original plants of the traditional Chinese medicine Murraya paniculata. Its dried leaves and leafy young branches are used medicinally. The name "Murraya paniculata" first appeared in the Ming Dynasty, and it was popularly known as "Nine-Mile Fragrance" in the Lingnan region due to its fragrant flowers. Botanists classify both Murraya paniculata and Murraya paniculata as belonging to the Murraya section of the Murraya genus in the Rutaceae family. Murraya paniculata is warm in nature, pungent and slightly bitter in taste, and has the effects of promoting qi circulation, relieving pain, and invigorating blood circulation. It is rich in coumarins, flavonoids, alkaloids, volatile oils, and other chemical components. Modern pharmacological studies show that Murraya paniculata has anti-inflammatory, analgesic, anti-tumor, hypoglycemic, insecticidal, antibacterial, osteoporosis prevention, antifertility, and antioxidant effects. Clinically, it is often used to treat inflammatory diseases such as chronic gastritis. Many commercially available traditional Chinese medicines, such as Sanjiu Weitai granules, Jiulong Weiyao, and Fuyinjie compound Huangsong lotion, all contain Murraya paniculata.

[0003] The stamens of Murraya paniculata (also known as Nine-mile Fragrance or Thousand-mile Fragrance) are severely degenerated, resulting in limited seed production. Therefore, seed propagation is difficult for large-scale, efficient cultivation. Plant tissue culture technology offers advantages such as a short growth cycle, high propagation rate, and convenient management, making it suitable for industrialized production and automated control. Therefore, plant tissue culture technology can better facilitate large-scale cultivation, selection of superior varieties, and rapid propagation of Murraya paniculata. While the explant-shoot-proliferation-rooting method can address the shortcomings of short roots or low rooting rates in Murraya paniculata tissue culture, it does not solve the problem of adventitious bud elongation.

[0004] In the tissue culture research of Murraya paniculata, there are problems such as slow elongation and poor elongation effect of adventitious buds during subculture, which seriously hinders the promotion and application of rapid propagation technology of Murraya paniculata through tissue culture. Summary of the Invention

[0005] One objective of this application is to provide a culture medium that promotes the elongation of adventitious buds in Murraya paniculata, thereby solving the problems of slow elongation and low effective bud rate of adventitious buds during subculturing.

[0006] Another objective of this application is to provide a method for promoting the elongation of adventitious buds in Murraya paniculata, thereby solving the problems of slow elongation and low effective bud rate of adventitious buds during the subculturing process.

[0007] Another objective of this application is to provide a method for cultivating Murraya paniculata tissue culture seedlings, thereby solving the problems of stunted growth and slow development of Murraya paniculata tissue culture seedlings.

[0008] In order to achieve these objectives and other advantages of this application, in a first aspect, this application provides a culture medium for promoting the elongation of adventitious shoots of Murraya paniculata, the culture medium for promoting the elongation of adventitious shoots of Murraya paniculata includes a shoot induction medium, a proliferation medium and an elongation medium.

[0009] The bud induction medium is prepared by adding 6-BA and IAA to the basal medium;

[0010] The proliferation medium is supplemented with 6-BA and IAA in the basal medium;

[0011] The elongation medium is a basal medium supplemented with 6-BA and IAA, or 6-BA, GA3 and NAA, or 6-BA and GA3.

[0012] In one optional embodiment, the amount of 6-BA added to the proliferation medium is 0.8 mg / L-2.4 mg / L, and the amount of IAA added is 0.1 mg / L-0.5 mg / L;

[0013] Preferably, the proliferation medium further includes KT, and the amount of KT added is 0.5 mg / L-1.0 mg / L;

[0014] More preferably, the amount of 6-BA added in the proliferation medium is 0.8 mg / L; the amount of KT added is 0.5 mg / L; and the amount of IAA added is 0.3 mg / L.

[0015] In one optional embodiment, the amount of 6-BA added to the bud induction medium is 0.6 mg / L-1.0 mg / L, and the amount of IAA added is 0.05 mg / L-0.15 mg / L.

[0016] In one optional embodiment, if 6-BA and IAA are added to the elongation medium, the amount of 6-BA added is 0.1 mg / L-0.3 mg / L, and the amount of IAA added is 0.01 mg / L-0.03 mg / L.

[0017] More preferably, the amount of 6-BA added is 0.2 mg / L, and the amount of IAA added is 0.02 mg / L.

[0018] In one optional embodiment, if 6-BA and GA3 are added to the elongation medium, the amount of 6-BA added is 0.1 mg / L-0.5 mg / L, and the amount of GA3 added is 0.4 mg / L-0.6 mg / L.

[0019] Preferably, the amount of 6-BA added is 0.5 mg / L or 0.3 mg / L, and the amount of GA3 added is 0.5 mg / L.

[0020] In one optional embodiment, if 6-BA, GA3, and NAA are added to the elongation medium, the amount of 6-BA added is 0.6 mg / L-1.0 mg / L, the amount of GA3 added is 0.4 mg / L-0.6 mg / L, and the amount of NAA added is 1-3 mg / L.

[0021] Preferably, the amount of 6-BA added is 0.8 mg / L, the amount of GA3 added is 0.5 mg / L, and the amount of NAA added is 2.0 mg / L.

[0022] In one optional embodiment, the pH of the culture medium for promoting the elongation of adventitious shoots of Murraya paniculata is adjusted to 5.4-6.2 before use;

[0023] The basal culture medium includes MS medium;

[0024] Preferably, the basal culture medium further includes sucrose and agar powder;

[0025] More preferably, the amount of sucrose added to the basal culture medium is 30 g / L, and the amount of agar powder added to the basal culture medium is 4.5 g / L.

[0026] Secondly, this application provides a method for promoting the elongation of adventitious buds in Murraya paniculata, comprising the following steps:

[0027] 1) Adventitious shoots of Murraya paniculata were placed in a proliferation medium for proliferation culture, and subculture was performed to obtain Murraya paniculata adventitious shoot clusters;

[0028] 2) The adventitious buds of Murraya paniculata were placed in an adventitious bud elongation medium to obtain elongated Murraya paniculata adventitious buds;

[0029] Preferably, the adventitious shoots of Murraya paniculata are obtained by inoculating Murraya paniculata explants into a shoot induction medium for induction culture.

[0030] In one optional implementation, the culture conditions for induction culture, proliferation culture, and elongation culture are as follows: culture temperature of 23℃-27℃, light intensity of 1500 lux-2000 lux, and light duration of 14-18 h / d.

[0031] Secondly, this application provides a method for cultivating Murraya paniculata tissue culture seedlings, comprising the following steps:

[0032] The adventitious buds or clusters of adventitious buds of Murraya paniculata obtained by the culture medium for promoting the elongation of adventitious buds of Murraya paniculata or by the method for promoting the elongation of adventitious buds of Murraya paniculata are inoculated into the rooting medium and cultured until roots are formed to obtain complete plants.

[0033] The rooting medium is based on 1 / 2 MS, agar powder and sucrose, with the addition of IBA, NAA and IAA.

[0034] The dosage of IBA in the rooting medium is 1.0 mg / L-2.0 mg / L, the dosage of NAA in the rooting medium is 1.0 mg / L-3.0 mg / L, and the dosage of IAA in the rooting medium is 0.5 mg / L-1.5 mg / L.

[0035] The amount of sucrose added to the basal culture medium is 30 g / L, and the amount of agar powder added to the basal culture medium is 4.5 g / L.

[0036] In one optional implementation, the rooting culture conditions are as follows: culture temperature of 23℃-27℃, light intensity of 1500 lux-2000 lux, and light duration of 14-18 h / d.

[0037] This application includes at least the following beneficial effects:

[0038] 1. This application provides a culture medium for promoting the elongation of adventitious shoots in Murraya paniculata, including a shoot induction medium, a proliferation medium, and an elongation medium. The shoot induction medium is prepared by adding 6-BA and IAA to the basal medium, and the proliferation medium is prepared by adding 6-BA and IAA to the basal medium. The elongation medium is prepared by adding 6-BA and IAA to the basal medium, or by adding 6-BA, GA3, and NAA, or by adding 6-BA and GA3. Using different plant growth regulators can effectively promote the elongation of adventitious shoots in Murraya paniculata, resulting in a large number of shoots and a high effective shoot rate. The cultured plants are robust and not stunted, effectively promoting the growth of adventitious shoots in Murraya paniculata, solving the technical problem of slow growth, and effectively shortening the culture cycle, thus facilitating better subsequent rooting culture of Murraya paniculata tissue culture seedlings.

[0039] 2. This application improves the effective bud rate by using a method to promote the elongation of adventitious buds of Murraya paniculata after 40 days of cultivation. This effectively promotes the growth of adventitious buds of Murraya paniculata and shortens the cultivation cycle, making it possible to rapidly propagate and cultivate Murraya paniculata tissue culture seedlings. Attached Figure Description

[0040] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0041] Figure 1 The aseptic adventitious buds of Murraya paniculata in Example 1;

[0042] Figure 2 The effect of different concentrations of 6-BA and IAA on the elongation of adventitious shoots of Murraya paniculata in Example 1 is shown. A to F correspond to 6-BA (0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L) and IAA (0.01 mg / L, 0.02 mg / L, 0.03 mg / L, 0.04 mg / L, 0.05 mg / L), respectively.

[0043] Figure 3 To compare the effects of 0.8 mg / L 6-BA and different concentrations of IAA on the elongation of adventitious shoots of Murraya paniculata in Example 1, A to E correspond to 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 3.0 mg / L, and 4.0 mg / L of IAA, respectively.

[0044] Figure 4 To compare the effects of 0.8 mg / L 6-BA and different concentrations of IBA on the elongation of adventitious shoots of Murraya paniculata in Example 2, A to E corresponded to 0.5, 1.0, 2.0, 3.0, and 4.0 mg / L IBA, respectively;

[0045] Figure 5 To compare the effects of 0.8 mg / L 6-BA and different concentrations of NAA on the elongation of adventitious shoots of Murraya paniculata in Example 3, A to E corresponded to 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 3.0 mg / L, and 4.0 mg / L NAA, respectively;

[0046] Figure 6 To compare the effects of different concentrations of 6-BA and NAA in Example 4 on the elongation of adventitious shoots of Murraya paniculata, A to F corresponded to 6-BA (0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L) and NAA (0.01 mg / L, 0.02 mg / L, 0.03 mg / L, 0.04 mg / L, 0.05 mg / L), respectively.

[0047] Figure 7 The effect of different concentrations of 6-BA and GA3 on the elongation of adventitious shoots of Murraya paniculata in Example 4 is shown in Figure 4. A is 0.1 mg / L 6-BA and 0.5 mg / L GA3; B is 0.3 mg / L 6-BA and 0.5 mg / L GA3; C is 0.5 mg / L 6-BA and 0.5 mg / L GA3; and D is 0.8 mg / L 6-BA, 0.5 mg / L GA3, and 2.0 mg / L NAA.

[0048] Figure 8 Adventitious roots induced from the adventitious buds of Murraya paniculata. Detailed Implementation

[0049] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0050] Existing technologies have disclosed rapid propagation methods for Murraya paniculata, including how to inoculate Murraya paniculata explants into a culture medium to promote proliferation and rooting. The proliferation medium increases the bud induction rate (number of adventitious buds), and then a rooting medium is used to obtain complete plants. However, the adventitious buds obtained in the bud proliferation medium are short and not robust, resulting in a high survival rate during later cultivation. In the cultivation of Murraya paniculata, it was found that taller adventitious buds are more likely to survive in later stages, and buds with a height ≥1.5cm all survived and grew robustly during later cultivation. Therefore, the elongation culture of Murraya paniculata adventitious buds is a key step in Murraya paniculata tissue culture technology and an important step required for subsequent rooting culture. Current research lacks studies on the elongation of Murraya paniculata adventitious buds.

[0051] In order to make the adventitious buds of Murraya paniculata grow longer and the effective bud rate higher, this application provides a culture medium for promoting the elongation of the adventitious buds of Murraya paniculata, which includes a bud induction culture medium, a proliferation culture medium and an elongation culture medium.

[0052] The bud induction medium is prepared by adding 6-BA and IAA to the basal medium;

[0053] The proliferation medium is supplemented with 6-BA and IAA in the basal medium;

[0054] The elongation medium is a basal medium supplemented with 6-BA and IAA, or 6-BA, GA3 and NAA, or 6-BA and GA3.

[0055] In some embodiments, the amount of 6-BA added to the proliferation medium is 0.8 mg / L-2.4 mg / L, and the amount of IAA added is 0.1 mg / L-0.5 mg / L;

[0056] That is, each liter of proliferation medium contains 0.8 mg-2.4 mg of 6-BA; each liter of proliferation medium contains 0.1 mg-0.5 mg of IAA.

[0057] Preferably, the proliferation medium further includes KT, and the amount of KT added is 0.5 mg / L-1.0 mg / L;

[0058] More preferably, the amount of 6-BA added in the proliferation medium is 0.8 mg / L; the amount of KT added is 0.5 mg / L; and the amount of IAA added is 0.3 mg / L.

[0059] In some embodiments, the amount of 6-BA added to the bud induction medium is 0.6 mg / L-1.0 mg / L, and the amount of IAA added is 0.05 mg / L-0.15 mg / L.

[0060] In some embodiments, if 6-BA and IAA are added to the elongation medium, the amount of 6-BA added is 0.1 mg / L-0.3 mg / L, and the amount of IAA added is 0.01 mg / L-0.03 mg / L.

[0061] More preferably, the amount of 6-BA added is 0.2 mg / L, and the amount of IAA added is 0.02 mg / L.

[0062] In some embodiments, if 6-BA and GA3 are added to the elongation medium, the amount of 6-BA added is 0.1 mg / L-0.5 mg / L, and the amount of GA3 added is 0.4 mg / L-0.6 mg / L.

[0063] Preferably, the amount of 6-BA added is 0.5 mg / L or 0.3 mg / L, and the amount of GA3 added is 0.5 mg / L.

[0064] In some embodiments, if 6-BA, GA3, and NAA are added to the elongation medium, the amount of 6-BA added is 0.6 mg / L-1.0 mg / L, the amount of GA3 added is 0.4 mg / L-0.6 mg / L, and the amount of NAA added is 1 mg / L-3 mg / L.

[0065] When the amount of GA3 added was 0.4 mg / L-0.6 mg / L, the difference in the elongation effect of adventitious shoots was not significant.

[0066] Preferably, the addition amount of 6-BA is 0.8 mg / L, the addition amount of GA3 is 0.5 mg / L, and the addition amount of NAA is 2 mg / L. The proliferation medium can promote adventitious bud propagation, facilitate subculture, and also strengthen seedlings.

[0067] In the proliferation medium of this application, the addition amount of 6-BA is 0.6 mg / L-1.0 mg / L (exemplary values ​​of 0.6 mg / L, 1.0 mg / L, 0.7 mg / L, and 0.8 mg / L can all be selected, and the effective budding rate of adventitious bud elongation is good with no significant difference), the addition amount of KT is 0.4 mg / L-0.6 mg / L (exemplary values ​​of 0.6 mg / L, 0.5 mg / L, and 0.4 mg / L can all be selected, and the effective budding rate of adventitious bud elongation is good with no significant difference), and the addition amount of IAA is 0.2 mg / L-0.4 mg / L (exemplary values ​​of 0.2 mg / L, 0.3 mg / L, and 0.4 mg / L can all be selected, and the effective budding rate of adventitious bud elongation is good with no significant difference).

[0068] The elongation medium primarily promotes the elongation of adventitious buds and also strengthens seedlings. This application incorporates 6-BA (a mitogen-6-BA) and auxins IAA, NAA, IBA, and / or GA3 into the elongation medium.

[0069] Experiments showed that adding 6-BA+IAA to the basal medium in the elongation medium had a good effect on the elongation of adventitious shoots. The use of a shoot induction medium increased the effective shoot rate.

[0070] The inventors further experimented with adding 6-BA+NAA, 6-BA+IBA, or 6-BA+GA3 to the elongation medium, which also showed good effects on the elongation of adventitious buds. Moreover, the effects of 6-BA+GA3 and 6-BA+IAA were better than those of adding 6-BA+IBA or 6-BA+NAA.

[0071] The inventors also tried adding 6-BA+GA3+NAA to the elongation medium and found that the elongation effect of adventitious shoots was not significantly different from that of adding 6-BA+GA3.

[0072] In some embodiments, the pH of the culture medium for promoting the elongation of adventitious shoots of Murraya paniculata is adjusted to 5.4-6.2 before use;

[0073] The basal culture medium includes MS medium;

[0074] Preferably, the basal culture medium further includes sucrose and agar powder;

[0075] More preferably, the amount of sucrose added to the basal culture medium is 20 g / L-40 g / L, and the amount of agar powder added to the basal culture medium is 3 g / L-6 g / L.

[0076] When the amount of sucrose added to the basal medium was 20 g / L-40 g / L and the amount of agar powder added to the basal medium was 3 g / L-6 g / L, the difference in the elongation effect of adventitious shoots was not significant.

[0077] More preferably, the amount of sucrose added to the basal culture medium is 30 g / L, and the amount of agar powder added to the basal culture medium is 4.5 g / L.

[0078] Secondly, this application provides a method for promoting the elongation of adventitious buds in Murraya paniculata, comprising the following steps:

[0079] 1) Adventitious shoots of Murraya paniculata were placed in a proliferation medium for proliferation culture, and subculture was performed to obtain Murraya paniculata adventitious shoot clusters;

[0080] 2) The adventitious buds of Murraya paniculata were placed in an adventitious bud elongation medium to obtain elongated Murraya paniculata adventitious buds;

[0081] Preferably, the adventitious shoots of Murraya paniculata are obtained by inoculating Murraya paniculata explants into a shoot induction medium for induction culture.

[0082] The culture conditions for induction culture, proliferation culture, and elongation culture were as follows: culture temperature 23℃-27℃, light intensity 1500lux-2000lux, and light duration 14-18h / d.

[0083] Secondly, this application provides a method for cultivating Murraya paniculata tissue culture seedlings, comprising the following steps:

[0084] The adventitious buds or clusters of adventitious buds of Murraya paniculata obtained by the culture medium for promoting the elongation of adventitious buds of Murraya paniculata or by the method for promoting the elongation of adventitious buds of Murraya paniculata are inoculated into the rooting medium and cultured until roots are formed to obtain complete plants.

[0085] The rooting medium is based on 1 / 2 MS, agar powder and sucrose, with added IBA, NAA and IAA;

[0086] 1 / 2 MS medium refers to MS medium with macroelements reduced to half of its original value;

[0087] The amount of IBA added is 1.0 mg / L-2.0 mg / L, the amount of NAA added is 1.0 mg / L-3.0 mg / L, and the amount of IAA added is 0.5 mg / L-1.5 mg / L.

[0088] Preferably, the amount of sucrose added to the basal culture medium is 30 g / L, and the amount of agar powder added to the basal culture medium is 4.5 g / L.

[0089] In some embodiments, the culture conditions for induction culture, proliferation culture, elongation culture or rooting culture are as follows: culture temperature of 23℃-27℃, light intensity of 1500lux-2000lux, and light duration of 14-18h / d.

[0090] This application takes into account the large number of adventitious buds in some treatment groups, making it difficult to measure the average height of all adventitious buds. Furthermore, considering that adventitious buds require a certain height for rooting culture, and that buds that are too short require artificial treatment, this application aims to ensure that all adventitious buds are at least 1.5 cm tall. Therefore, the effective bud rate is selected as the indicator of elongation in different treatment groups. Effective bud rate = (Number of seedlings with adventitious bud height ≥ 1.5 cm / Total number of adventitious buds) × 100%.

[0091] The specific culture media and hormones involved in this application are as follows: MS medium and 1 / 2 MS medium were purchased from Beijing Combes Technology Co., Ltd.; plant hormones: 6-BA refers to 6-benzylaminopurine, KT refers to 6-glycosylaminopurine, IAA refers to indole-3-acetic acid; IBA refers to indolebutyric acid, NAA refers to α-naphthaleneacetic acid, and GA3 refers to gibberellin, all of which were purchased from Combes Technology Co., Ltd.

[0092] The formulations of MS medium and 1 / 2 MS medium involved in this application are shown in Table 1:

[0093] Table 1 Formulations for 1 / 2MS and MS Media

[0094]

[0095]

[0096] Example 1

[0097] A method for promoting the elongation of adventitious buds in Murraya paniculata includes the following steps:

[0098] Step 1: Take 2-3cm stem segments of Murraya paniculata with 1-2 axillary buds as explants. Clean them with a soft brush, then rinse them thoroughly with tap water. Place the material in a beaker, transfer it to a clean bench, treat with 75% ethanol solution for 30 seconds, then with 0.1% HgCl2 disinfectant for 8 minutes. Finally, rinse four times with sterile water, blot dry with sterile filter paper, and inoculate into bud induction medium for 30 days. Use MS medium as the basal medium, supplemented with 0.8 mg / L 6-BA and 0.1 mg / L IAA to form adventitious buds. Figure 1 .

[0099] Step 2: Prepare the adventitious shoot proliferation medium for Murraya paniculata using MS medium, 6-BA, KT, and IAA, as follows:

[0100] L9 (3) was designed using 6-BA (0.8, 1.6, 2.4 mg / L), KT (0, 0.5, 1.0 mg / L), and IAA (0.1, 0.3, 0.5 mg / L). 3An orthogonal experiment was conducted to investigate the optimal scheme for adventitious shoot proliferation. Each treatment was inoculated with 20 shoots and cultured into explants, repeated three times. The shoot proliferation rate was calculated after 40 days [shoot proliferation rate = (number of shoots after 40 days - number of shoots at inoculation) / number of shoots at inoculation]. The effects of different treatment combinations on the adventitious shoot proliferation results are shown in Table 2 below.

[0101] Table 2 Results of the orthogonal experiment on the propagation culture of adventitious buds of Murraya paniculata

[0102]

[0103] Note: Different lowercase letters after the data in the same column indicate significant differences according to Duncan's test (P<0.05).

[0104] Table 3. Duncan's test for 6-BA, IAA, and KT concentration levels.

[0105]

[0106] Note: Different lowercase letters after the data in the same column indicate significant differences according to Duncan's test (P<0.05).

[0107] Table 2 shows that, based on the range of bud proliferation rates, 6-BA is the most important factor in the proliferation of adventitious buds of Murraya paniculata, followed by KT and IAA. The optimal combination is experiment number 2, meaning the best medium formula for adventitious bud proliferation of Murraya paniculata is MS + 6-BA 0.8 mg / L + IAA 0.3 mg / L + KT 0.5 mg / L. Combined with the analysis of variance results, 6-BA, KT, and IAA all have significant effects on the proliferation of adventitious buds of Murraya paniculata (P < 0.05). Furthermore, Duncan's test was performed on the three concentrations of 6-BA, KT, and IAA. Table 3 shows that the levels with the greatest impact on the proliferation of adventitious buds of Murraya paniculata are 6-BA level 1 (0.8 mg / L), IAA levels 1 and 2 (0.1 and 0.3 mg / L), and KT levels 1 and 2 (0 and 0.5 mg / L). Based on the above results and the growth status of the buds, the optimal culture medium for adventitious bud proliferation is MS + 6-BA 0.8 mg / L + IAA 0.3 mg / L + KT 0.5 mg / L.

[0108] The addition amount of 6-BA was 0.8 mg / L; the addition amount of KT was 0.5 mg / L; and the addition amount of IAA was 0.3 mg / L.

[0109] Step 3: Prepare adventitious shoot elongation medium using MS medium, 6-BA and IAA, with 6-BA added at 0.1-0.5 mg / L and IAA added at 0.01-0.05 mg / L, as shown in Table 4.

[0110] Step 4: Take 20 adventitious shoot explants of Murraya paniculata and place them in proliferation medium (MS + 6-BA 0.8 mg / L + IAA 0.3 mg / L + KT 0.5 mg / L) to subculture and obtain Murraya paniculata adventitious shoot clusters.

[0111] Step 5: Take four clusters of Murraya paniculata buds of similar size and growth and place them in elongation medium for 40 days. The buds will elongate significantly. The fresh weight and effective bud rate of the Murraya paniculata bud clusters will be recorded.

[0112] In addition to the above-mentioned components, all the above-mentioned culture media were supplemented with 30 g / L sucrose and 4.5 g / L agar powder, and the pH value of the culture media was adjusted to 5.8.

[0113] The culture conditions for induction culture, proliferation culture or elongation culture are as follows: temperature 25±2℃, light intensity 1500-2000 lux, and photoperiod 16h / d.

[0114] Table 4 shows the results of adventitious bud elongation of Murraya paniculata after 40 days in Example 1.

[0115]

[0116] Note: Different lowercase letters after the data in the same column indicate significant differences according to Duncan's test (P<0.05).

[0117] Table 4 shows that different concentrations of 6-BA and IAA significantly affected the effective budding rate of *Murraya paniculata* adventitious buds. The effective budding rates were higher when the concentrations of 6-BA and IAA were 0.1 mg / L-0.4 mg / L and 0.01 mg / L-0.04 mg / L, respectively; the highest effective budding rate (63.25%) was achieved when the concentrations of 6-BA and IAA were 0.2 mg / L and 0.02 mg / L, respectively. Figure 2 .

[0118] Furthermore, a method for cultivating Murraya paniculata tissue culture seedlings also includes:

[0119] Step Six: Inoculate the elongated adventitious shoots into rooting medium for 90 days. The rooting medium used was 1 / 2 MS as the basal medium, supplemented with 1.0 mg / L IBA, 1.5 mg / L NAA, and 1.0 mg / L IAA. Statistical analysis showed that the adventitious root induction rate was 34.72%, with an average of 2.63 roots per shoot.

[0120] In addition to the above-mentioned components, the rooting medium was supplemented with 30 g / L sucrose and 4.5 g / L agar powder, and the pH of the medium was adjusted to 5.8.

[0121] The conditions for rooting culture were: temperature 25±2℃, light intensity 1500-2000 lux, and photoperiod 16h / d.

[0122] Comparative Example 1

[0123] A method for promoting the elongation of adventitious buds in Murraya paniculata includes the following steps:

[0124] Take 2-3cm stem segments of Murraya paniculata with 1-2 axillary buds as explants. Clean them with a soft brush, then rinse them with tap water. Place the material in a beaker, transfer it to a clean bench, treat it with 75% ethanol solution for 30 seconds, treat it with 0.1% HgCl2 disinfectant solution for 8 minutes, and finally rinse it 4 times with sterile water. After drying with sterile filter paper, inoculate it into MS medium for culture.

[0125] Step 1: Prepare the adventitious shoot proliferation medium of Murraya paniculata using MS medium, 6-BA, KT and IAA as follows: 6-BA 0.8 mg / L + IAA 0.3 mg / L + KT 0.5 mg / L.

[0126] Step 2: Prepare adventitious shoot elongation medium using MS medium, 6-BA and IAA, where the amount of 6-BA added is 0.8 mg / L; the concentration of IAA is 0.5 mg / L-4.0 mg / L, as shown in Table 4.

[0127] Step 3: Take 20 adventitious bud explants of Murraya paniculata and place them in a proliferation medium to obtain Murraya paniculata adventitious bud clusters through subculture.

[0128] Step 4: Take adventitious bud clusters of Murraya paniculata with similar size and growth and place them in elongation medium for 40 days to allow the buds to elongate significantly. Calculate the fresh weight and effective bud rate of the Murraya paniculata adventitious bud clusters.

[0129] In addition to the above-mentioned components, all the above-mentioned culture media were supplemented with 30 g / L sucrose and 4.5 g / L agar powder, and the pH value of the culture media was adjusted to 5.8.

[0130] The culture conditions for induction culture, proliferation culture or elongation culture are as follows: temperature 25±2℃, light intensity 1500-2000 lux, and photoperiod 16h / d.

[0131] Table 5 shows the results of adventitious bud elongation in Comparative Example 1 after 40 days.

[0132]

[0133] Note: Different lowercase letters after the data in the same column indicate significant differences according to Duncan's test (P<0.05).

[0134] Table 5 shows that different concentrations of IAA did not significantly affect the elongation of adventitious shoots in Murraya paniculata. The highest effective shoot rate (25.44%) was achieved when the concentration of 6-BA was 0.8 mg / L and the concentration of IAA was 0.5 mg / L. Figure 3 .

[0135] A method for cultivating Murraya paniculata tissue culture seedlings also includes:

[0136] Step 6: Inoculate the elongated adventitious buds into the rooting medium and culture for 90 days until roots are formed to obtain a complete plant.

[0137] The rooting medium used was 1 / 2 MS medium as the basal medium, supplemented with 0.5 mg / L NAA. The adventitious root induction rate was 23.33%, and the average number of roots was 1.17.

[0138] In addition to the above-mentioned components, the rooting medium was supplemented with 30 g / L sucrose and 4.5 g / L agar powder, and the pH of the medium was adjusted to 5.8.

[0139] The rooting culture temperature was 25±2℃, the light intensity was 1500-2000 lux, and the photoperiod was 16h / d.

[0140] In both Example 1 and Comparative Example 1, the elongation medium was a basal medium supplemented with 6-BA and IAA. Example 1 also added a bud induction culture step. Through comparative experiments, it was found that using the bud induction medium of this application to culture explants not only helps to improve the budding rate of explants, but also effectively improves the effective budding rate of adventitious buds, while greatly reducing the amount of 6-BA and IAA used.

[0141] Example 2

[0142] The method in this embodiment is basically the same as that in Example 1, except that 0.6 mg / L of 6-BA and 0.15 mg / L of IAA are added to the bud induction medium.

[0143] Example 3

[0144] The method in this embodiment is basically the same as that in Example 1, except that 1.0 mg / L of 6-BA and 0.05 mg / L of IAA are added to the bud induction medium.

[0145] Comparative results from Examples 1-3 showed that adding different concentrations of 6-BA and IAA to the bud induction medium did not significantly affect the elongation of the number of adventitious buds of Murraya paniculata. The effective budding rate of Murraya paniculata was highest when the concentration of 6-BA was 0.8 mg / L and the concentration of IAA was 0.1 mg / L.

[0146] Comparative Example 2

[0147] A method for promoting the elongation of adventitious buds in Murraya paniculata includes the following steps:

[0148] Step 1: Take 2-3cm stem segments of Murraya paniculata with 1-2 axillary buds as explants. Clean them with a soft brush, then rinse them with tap water. Place the material in a beaker, transfer it to a clean bench, treat it with 75% ethanol solution for 30 seconds, then treat it with 0.1% HgCl2 disinfectant solution for 8 minutes, and finally rinse it 4 times with sterile water. After drying with sterile filter paper, inoculate it into bud induction medium for 30 days. Use MS medium as the basic medium, with 0.8mg / L 6-BA and 0.1mg / L IAA added.

[0149] Step 2: Prepare a medium for the proliferation of adventitious shoots of Murraya paniculata using MS medium, 6-BA, KT and IAA, with 6-BA added at 0.8 mg / L, KT added at 0.5 mg / L and IAA added at 0.3 mg / L.

[0150] Step 3: Prepare adventitious shoot elongation medium using MS medium, 6-BA and IBA, with 6-BA added at a concentration of 0.8 mg / L and IBA added at a concentration of 0.5 mg / L to 4.0 mg / L, as shown in Table 6.

[0151] Step 4: Take 20 adventitious shoot explants of Murraya paniculata and place them in a proliferation medium to subculture and obtain Murraya paniculata adventitious shoot clusters.

[0152] Step 5: Take four clusters of Murraya paniculata buds of similar size and growth and place them in elongation medium for 40 days. The buds will elongate significantly. The fresh weight and effective bud rate of the Murraya paniculata bud clusters will be recorded.

[0153] In addition to the above-mentioned components, all the above-mentioned culture media were supplemented with 30 g / L sucrose and 4.5 g / L agar powder, and the pH value of the culture media was adjusted to 5.8.

[0154] The culture conditions for induction culture, proliferation culture or elongation culture are as follows: temperature 25±2℃, light intensity 1500~2000 lux, and photoperiod 16h / d.

[0155] Table 6 shows the results of adventitious bud elongation in Comparative Example 2 after 40 days.

[0156]

[0157] Note: Different lowercase letters after the data in the same column indicate significant differences according to Duncan's test (P<0.05).

[0158] In this embodiment, the elongation medium was prepared by adding 6-BA and IBA to the basal medium. Table 6 shows that different concentrations of IBA affected the effective budding rate of *Murraya paniculata* adventitious buds. As the IBA concentration increased, the effective budding rate first increased and then decreased. The effective budding rate was higher when the IBA concentration was between 1.0 mg / L and 4.0 mg / L, and the highest effective budding rate (43.86%) was observed at an IBA concentration of 3.0 mg / L. Figure 4 .

[0159] Furthermore, a method for cultivating Murraya paniculata tissue culture seedlings also includes:

[0160] Step Six: Inoculate the elongated adventitious shoots into rooting medium for 90 days. The medium used is 1 / 2 MS as the basal medium, supplemented with 1.0 mg / L IBA, 2.0 mg / L NAA, 0.5 mg / L IAA, and 0.1 mg / L KT. The adventitious root induction rate was 25.00%, with an average of 1.33 roots per shoot.

[0161] In addition to the above-mentioned components, the rooting medium was supplemented with 30 g / L sucrose and 4.5 g / L agar powder, and the pH of the medium was adjusted to 5.8.

[0162] The conditions for rooting culture were: temperature 25±2℃, light intensity 1500-2000 lux, and photoperiod 16h / d.

[0163] Comparative Example 3

[0164] A method for promoting the elongation of adventitious buds in Murraya paniculata includes the following steps:

[0165] Step 1: Take 2-3cm stem segments of Murraya paniculata with 1-2 axillary buds as explants. Clean them with a soft brush, then rinse them with tap water. Place the material in a beaker, transfer it to a clean bench, treat it with 75% ethanol solution for 30 seconds, then treat it with 0.1% HgCl2 disinfectant solution for 8 minutes, and finally rinse it 4 times with sterile water. After drying with sterile filter paper, inoculate it into bud induction medium for 30 days. Use MS medium as the basic medium, with 0.8mg / L 6-BA and 0.1mg / L IAA added.

[0166] Step 2: Prepare a medium for the proliferation of adventitious shoots of Murraya paniculata using MS medium, 6-BA, KT and IAA, with 6-BA added at 0.8 mg / L, KT added at 0.5 mg / L and IAA added at 0.3 mg / L.

[0167] Step 3: Prepare adventitious shoot elongation medium using MS medium, 6-BA and NAA, with 6-BA added at 0.8 mg / L and NAA added at 0.5-4.0 mg / L, as shown in Table 7.

[0168] Step 4: Take 20 adventitious shoot explants of Murraya paniculata and place them in a proliferation medium to subculture and obtain Murraya paniculata adventitious shoot clusters.

[0169] Step 5: Take four clusters of Murraya paniculata buds of similar size and growth and place them in elongation medium for 40 days. The buds will elongate significantly. The fresh weight and effective bud rate of the Murraya paniculata bud clusters will be recorded.

[0170] In addition to the above-mentioned components, all the above-mentioned culture media were supplemented with 30 g / L sucrose and 4.5 g / L agar powder, and the pH value of the culture media was adjusted to 5.8.

[0171] The culture conditions for induction culture, proliferation culture or elongation culture are as follows: temperature 25±2℃, light intensity 1500~2000 lux, and photoperiod 16h / d.

[0172] Table 7 shows the results of the adventitious bud elongation of Murraya paniculata after 40 days in Comparative Example 3.

[0173]

[0174] Note: Different lowercase letters after the data in the same column indicate significant differences according to Duncan's test (P<0.05).

[0175] In this embodiment, the elongation medium was prepared by adding 6-BA and NAA to the basal medium. Table 7 shows that different concentrations of NAA affected the effective budding rate of *Murraya paniculata* adventitious shoots. As the 6-BA concentration increased, the effective budding rate first increased and then decreased. The effective budding rate was higher when the NAA concentration was between 2.0 mg / L and 4.0 mg / L. The highest effective budding rate (46.30%) was achieved when the NAA concentration was 2.0 mg / L. Figure 5 .

[0176] Furthermore, a method for cultivating Murraya paniculata tissue culture seedlings also includes:

[0177] Step Six: Inoculate the elongated adventitious shoots into rooting medium for 90 days. The medium used is 1 / 2 MS as the basal medium, supplemented with 0.5 mg / L IBA, 2.0 mg / L NAA, 1.5 mg / L IAA, and 0.5 mg / L KT. The adventitious root induction rate was 27.78%, with an average of 1.70 roots per shoot.

[0178] In addition to the above-mentioned components, the rooting medium was supplemented with 30 g / L sucrose and 4.5 g / L agar powder, and the pH of the medium was adjusted to 5.8.

[0179] The rooting culture conditions were as follows: temperature 25±2℃, light intensity 1500-2000 lux, and photoperiod 16h / d.

[0180] Comparisons in Example 1 and Comparative Examples 2-3 show that adding 6-BA+NAA to the elongation medium is more effective than adding 6-BA+IBA, but far less effective than adding 6-BA+IAA. Therefore, adjusting the ratio of 6-BA to NAA is considered to achieve better results.

[0181] Comparative Example 4: Effects of different concentrations of 6-BA and NAA on the elongation of adventitious shoots of Murraya paniculata

[0182] A method for promoting the elongation of adventitious buds in Murraya paniculata includes the following steps:

[0183] Step 1: Take 2-3cm stem segments of Murraya paniculata with 1-2 axillary buds as explants. Clean them with a soft brush, then rinse them with tap water. Place the material in a beaker, transfer it to a clean bench, treat it with 75% ethanol solution for 30 seconds, then treat it with 0.1% HgCl2 disinfectant solution for 8 minutes, and finally rinse it 4 times with sterile water. After drying with sterile filter paper, inoculate it into bud induction medium for 30 days. Use MS medium as the basic medium, with 0.8mg / L 6-BA and 0.1mg / L IAA added.

[0184] Step 2: Prepare a medium for the proliferation of adventitious shoots of Murraya paniculata using MS medium, 6-BA, KT and IAA, with 6-BA added at 0.8 mg / L, KT added at 0.5 mg / L and IAA added at 0.3 mg / L.

[0185] Step 3: Prepare adventitious shoot elongation medium using MS medium, 6-BA and IAA, where the amount of 6-BA added is 0.1-0.5 g / L and the amount of NAA added is 0.01-0.05 mg / L, as shown in Table 8.

[0186] Step 4: Take 20 adventitious shoot explants of Murraya paniculata and place them in a proliferation medium to subculture and obtain Murraya paniculata adventitious shoot clusters.

[0187] Step 5: Take four clusters of Murraya paniculata buds of similar size and growth and place them in elongation medium for 40 days. The buds will elongate significantly. The fresh weight and effective bud rate of the Murraya paniculata bud clusters will be recorded.

[0188] In addition to the above-mentioned components, all the above-mentioned culture media were supplemented with 30 g / L sucrose and 4.5 g / L agar powder, and the pH value of the culture media was adjusted to 5.8.

[0189] The culture conditions for induction culture, proliferation culture or elongation culture are as follows: temperature 25±2℃, light intensity 1500-2000 lux, and photoperiod 16h / d.

[0190] Table 8 shows the results of adventitious bud elongation in Comparative Example 4 after 40 days.

[0191]

[0192]

[0193] Note: Different lowercase letters after the data in the same column indicate significant differences according to Duncan's test (P<0.05).

[0194] As shown in Table 8, the highest effective germination rate (45.79%) was achieved when the 6-BA concentration was 0.1 mg / L and the NAA concentration was 0.01 mg / L. Figure 6 .

[0195] Furthermore, a method for cultivating Murraya paniculata tissue culture seedlings also includes:

[0196] Step Six: Inoculate the elongated adventitious shoots into rooting medium for 90 days. The medium used was 1 / 2 MS as the basal medium, supplemented with 1.0 mg / L IBA, 1.0 mg / L NAA, 1.0 mg / L IAA, and 0.5 mg / L KT. The adventitious root induction rate was 37.50%, with an average of 2.00 roots per shoot.

[0197] In addition to the above-mentioned components, the rooting medium was supplemented with 30 g / L sucrose and 4.5 g / L agar powder, and the pH of the medium was adjusted to 5.8.

[0198] The rooting culture conditions were as follows: temperature 25±2℃, light intensity 1500-2000 lux, and photoperiod 16h / d. Comparison of Example 1 and Comparative Example 4 showed that adjusting the ratio of 6-BA to NAA did not result in a higher effective budding rate compared to adding 6-BA+IAA.

[0199] Example 4

[0200] A method for promoting the elongation of adventitious buds in Murraya paniculata includes the following steps:

[0201] Step 1: Take 2-3cm stem segments of Murraya paniculata with 1-2 axillary buds as explants. Clean them with a soft brush, then rinse them with tap water. Place the material in a beaker, transfer it to a clean bench, treat it with 75% ethanol solution for 30 seconds, then treat it with 0.1% HgCl2 disinfectant solution for 8 minutes, and finally rinse it 4 times with sterile water. After drying with sterile filter paper, inoculate it into bud induction medium for 30 days. Use MS medium as the basic medium, with 0.8mg / L 6-BA and 0.1mg / L IAA added.

[0202] Step 2: Prepare a medium for the proliferation of adventitious shoots of Murraya paniculata using MS medium, 6-BA, KT and IAA, with 6-BA added at 0.8 mg / L, KT added at 0.5 mg / L and IAA added at 0.3 mg / L.

[0203] Step 3: Prepare adventitious shoot elongation medium using MS medium, 6-BA, NAA and GA3. The amount of 6-BA added is 0.1-0.8 g / L; the amount of GA3 added is 0.5 mg / L; and the amount of NAA added is 0 or 2.0 mg / L, as shown in Table 9.

[0204] Step 4: Place the adventitious buds of Murraya paniculata in a proliferation medium and subculture to obtain Murraya paniculata adventitious bud clusters.

[0205] Step 5: Take adventitious buds of similar size and growth from the adventitious bud clusters in Step 4 (remove those that are too short or too long) and place them in the elongation medium in Step 3 for 40 days. Take 4 clusters per bottle to allow the buds to elongate significantly. Calculate the fresh weight and effective bud rate of the adventitious bud clusters of Murraya paniculata.

[0206] In addition to the above-mentioned components, all the above-mentioned culture media were supplemented with 30 g / L sucrose and 4.5 g / L agar powder, and the pH value of the culture media was adjusted to 5.8.

[0207] The culture conditions for induction culture, proliferation culture or elongation culture are as follows: temperature 25±2℃, light intensity 1500-2000 lux, and photoperiod 16h / d.

[0208] Table 9 shows the results of adventitious bud elongation of Murraya paniculata after 40 days in Example 4.

[0209]

[0210] Note: Different lowercase letters after the data in the same column indicate significant differences according to Duncan's test (P<0.05).

[0211] As shown in Table 9, different ratios of plant growth regulators affected the effective bud rate of *Murraya paniculata* adventitious buds. Adding 6-BA + GA3 significantly promoted the elongation of *Murraya paniculata* adventitious buds. In this example, there was no significant difference in the effective bud rate among the treatment groups. When 6-BA was 0.5 mg / L and GA3 was 0.5 mg / L, the adventitious bud elongation effect was better, with an effective bud rate of 69.53%. The plants were robust, and more effective buds were obtained within the same culture time, effectively shortening the production cycle to a certain extent. Figure 7 In practical applications, cost factors such as plant hormones can be taken into account. 0.5 mg / L GA3 and 0.3 mg / L 6-BA can be added to MS medium to promote the elongation of adventitious shoots of Murraya paniculata.

[0212] Furthermore, a method for cultivating Murraya paniculata tissue culture seedlings also includes:

[0213] Step Six: Inoculate the elongated adventitious shoots into rooting medium for 90 days. The medium used is 1 / 2 MS as the basal medium, supplemented with 1.5 mg / L IBA, 2.0 mg / L NAA, and 1.0 mg / L IAA. The adventitious root induction rate was 58.33%, with an average of 2.95 roots per shoot. Figure 8 Adventitious roots induced from the adventitious buds of Murraya paniculata.

[0214] In addition to the above-mentioned components, the rooting medium was supplemented with 30 g / L sucrose and 4.5 g / L agar powder, and the pH of the medium was adjusted to 5.8.

[0215] The conditions for rooting culture were: temperature 25±2℃, light intensity 1500-2000 lux, and photoperiod 16h / d.

[0216] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this application.

Claims

1. A method of promoting the elongation of Plumeria alba adventitious shoots, characterized by, The steps are as follows: The culture medium for promoting the elongation of adventitious shoots of Murraya paniculata includes a shoot induction medium, a proliferation medium, and an elongation medium. 1) Adventitious buds of Murraya paniculata were obtained by inoculating Murraya paniculata stem segments with 1-2 axillary buds into a bud induction medium for induction culture; the bud induction medium consisted of MS basal medium, 6-BA and IAA; the amount of 6-BA added to the bud induction medium was 0.6 mg / L-1.0 mg / L, and the amount of IAA added was 0.05 mg / L-0.15 mg / L; The adventitious buds of Murraya paniculata were placed in a proliferation medium for proliferation culture, and the adventitious bud clusters of Murraya paniculata were obtained by subculture. The proliferation medium consisted of MS basal medium, 0.8 mg / L 6-BA, 0.1 mg / L-0.3 mg / L L1AA, and KT 0-0.5 mg / L. 2) The adventitious buds of Murraya paniculata were placed in an adventitious bud elongation medium to obtain elongated Murraya paniculata adventitious buds; The elongation medium consists of MS basal medium, 6-BA 0.1 mg / L-0.3 mg / L and IAA 0.01 mg / L-0.03 mg / L, or MS basal medium, 6-BA 0.6 mg / L-1.0 mg / L, GA3 0.4 mg / L-0.6 mg / L and NAA 1 mg / L-3 mg / L, or MS basal medium, 6-BA 0.1 mg / L-0.5 mg / L and GA3 0.4 mg / L-0.6 mg / L. The culture conditions for induction culture, proliferation culture or elongation culture are as follows: culture temperature 23℃-27℃, light intensity 1500 lux-2000 lux, and light duration 14-18h / d.

2. The method according to claim 1, characterized in that, The elongation medium consisted of MS basal medium, 6-BA 0.2 mg / L and IAA 0.02 mg / L.

3. The method according to claim 1 or 2, characterized in that, The elongation medium consists of MS basal medium, 6-BA 0.5 mg / L and GA 3 0.5 mg / L, or the elongation medium consists of MS basal medium, 6-BA 0.3 mg / L and GA 3 0.5 mg / L.

4. The method according to claim 1, characterized in that, The elongation medium consists of MS basal medium, 0.8 mg / L 6-BA, 0.5 mg / L GA3 and 2 mg / L NAA.

5. The method according to claim 1, characterized in that, The pH of the culture medium used to promote the elongation of adventitious shoots of Murraya paniculata was adjusted to 5.4-6.2 before use. The basal culture medium also includes sucrose and agar powder; The amount of sucrose added to the basal culture medium is 30 g / L, and the amount of agar powder added to the basal culture medium is 4.5 g / L.

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