Medium for tissue culture of Melilotus chinensis and tissue culture method of Melilotus chinensis

Abstract

The invention discloses a culture medium for tissue culture of Melilotus chinensis and a tissue culture method of Melilotus chinensis, and belongs to the technical field of plant tissue culture. The culture medium for tissue culture of Melilotus chinensis of the invention comprises a callus induction culture medium, a redifferentiation bud induction culture medium, and a rooting culture medium. The invention determines the components of the callus induction culture medium, the redifferentiation bud induction culture medium, and the rooting culture medium of Melilotus chinensis explants by studying the culture medium components of callus induction, redifferentiation bud induction, and rooting induction of Melilotus chinensis explants, and successfully establishes an efficient tissue culture system of Melilotus chinensis. It can not only overcome the limitations of traditional breeding methods and improve breeding efficiency, but also lay a foundation for the subsequent genetic breeding of Melilotus chinensis with good stress resistance and high grass yield using genetic engineering.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a culture medium for Melilotus odoratus tissue culture and a tissue culture method of Melilotus odoratus. Background Art

[0002] Melilotus Miller is an annual or perennial herbaceous plant of the Leguminosae family. Among them, the medicinal Melilotus officinalis Pall., also known as yellow sweet osmanthus and sweet osmanthus, has strong resistance to barrenness, drought and alkali. It is often used as a biological desalinator to improve saline-alkali land. It can effectively absorb salt and alkali in the soil, improve soil structure and maintain water and soil.

[0003] Soil salinization is a common problem faced by countries around the world today. Solving the problem of soil salinization is a major and challenging issue for future agricultural development. In recent years, vegetation restoration has received increasing attention. Melilotus officinalis is a commonly used plant for improving saline-alkali land. The establishment of a tissue culture system for Melilotus officinalis can improve the success rate and reproduction coefficient of reproduction, and can also provide a theoretical basis and technical support for the genetic breeding of Melilotus officinalis for salt-alkali tolerance using genetic transformation technology.

[0004] Melilotus can be generally divided into white Melilotus and yellow Melilotus. At present, the research on the establishment of Melilotus tissue culture system mainly focuses on white Melilotus, and there are few reports on the establishment of a complete and efficient white Melilotus tissue culture system; Yellow Melilotus is better than White Melilotus in terms of resistance to frost, drought and disease, and its grass yield is lower than that of White Melilotus. Therefore, the establishment of a complete and efficient yellow Melilotus tissue culture system can not only overcome the limitations of traditional breeding methods and improve breeding efficiency, but also has broad application prospects in agricultural production, germplasm resource protection and new variety breeding. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a culture medium for Melilotus chinensis tissue culture and a tissue culture method for Melilotus chinensis.

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

[0007] A culture medium for Melilotus chinensis tissue culture, the culture medium comprising a callus induction medium, a redifferentiation bud induction medium, and a rooting medium;

[0008] The callus induction medium comprises:

[0009] MS medium + 0.5 mg / L 2,4-D + 0.25 mg / L 6-BA + 0.25 mg / L NAA + 30 g / L sucrose + 8 g / L agar, pH 5.8-6.0.

[0010] On the basis of the above scheme, the composition of the redifferentiation budding induction medium is: MS medium without organic matter + B5 medium organic matter + 1 mg / L 6-BA + 0.2 mg / L IAA + 10 mL / L pure coconut water + 30 g / L sucrose + 8 g / L agar, with a pH of 5.8-6.0.

[0011] On the basis of the above scheme, the rooting medium is composed of any one of the following culture media:

[0012] Rooting medium 1: 1 / 2MS medium + 0.2mg / L IBA + 15g / L sucrose + 5g / L agar, pH 5.8-6.0;

[0013] Rooting medium 2: 1 / 2MS medium + 0.2 mg / L NAA + 15 g / L sucrose + 5 g / L agar, pH 5.8-6.0.

[0014] Application of the above-mentioned culture medium for Melilotus tissue culture in Melilotus tissue culture.

[0015] Based on the above scheme, the Melilotus odoratus is Melilotus odoratus.

[0016] A tissue culture method of Melilotus officinalis, comprising the following steps:

[0017] (1) taking a Melilotus officinalis explant and inoculating it into a callus induction medium to induce callus;

[0018] (2) Selecting the light yellow callus tissue induced in step (1) with a clumping structure and larger callus pieces and inoculating them onto a redifferentiation sprouting induction medium to induce the formation of callus differentiation sprouts.

[0019] (3) The buds induced in step (2) are placed in MS solid medium without any hormones for cultivation, and when they grow to 3-5 cm, they are transferred to rooting medium to induce rooting;

[0020] The callus induction medium comprises:

[0021] MS medium + 0.5 mg / L 2,4-D + 0.25 mg / L 6-BA + 0.25 mg / L NAA + 30 g / L sucrose + 8 g / L agar, pH 5.8-6.0;

[0022] The redifferentiation sprout induction medium comprises: MS medium without organic matter + B5 medium organic matter + 1 mg / L 6-BA + 0.2 mg / L IAA + 10 mL / L pure coconut water + 30 g / L sucrose + 8 g / L agar, with a pH of 5.8-6.0;

[0023] The rooting medium is composed of any one of the following culture media:

[0024] Rooting medium 1: 1 / 2MS medium + 0.2mg / L IBA + 15g / L sucrose + 5g / L agar, pH 5.8-6.0;

[0025] Rooting medium 2: 1 / 2MS medium + 0.2 mg / L NAA + 15 g / L sucrose + 5 g / L agar, pH 5.8-6.0.

[0026] On the basis of the above scheme, the Melilotus explant is the hypocotyl, radicle or leaf of the Melilotus sterile seedling. Preferably, the Melilotus explant is the hypocotyl of the Melilotus sterile seedling.

[0027] Based on the above scheme, the Melilotus odoratus is Melilotus odoratus.

[0028] On the basis of the above scheme, the callus induction conditions in step (1) are: dark / light time 8 / 16h, temperature 22±2°C, air humidity 50-70%, light intensity 1000-2000lx, and culture for 14-28 days;

[0029] The conditions for inducing callus differentiation and bud formation in step (2) are: darkness / light time 8 / 16h, temperature 24±2°C, air humidity 50-70%, light intensity 2000-3000lx, and culture for 14-28 days;

[0030] The rooting induction conditions in step (3) are: darkness / light time 8 / 16h, temperature 24±2°C, air humidity 50-70%, light intensity 2000-3000lx, and cultivation for 14-28 days.

[0031] Advantages of the technical solution of the present invention

[0032] The present invention determines the components of the callus induction medium, the redifferentiation bud induction medium and the rooting medium of the Melilotus explant by studying the culture medium components of the Melilotus explant callus induction medium, the redifferentiation bud induction medium and the rooting medium, and successfully establishes an efficient tissue culture system of Melilotus chrysantha. It can not only overcome the limitations of traditional breeding methods and improve breeding efficiency, but also lay a foundation for the subsequent genetic breeding of Melilotus chrysantha with good stress resistance and high grass yield by using genetic engineering.

[0033] The high-efficiency tissue culture system of Melilotus luteus provided by the invention is used for tissue culture of Melilotus luteus and has broad application prospects in agricultural production, germplasm resource protection, new variety breeding and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Diagrams of callus induction using different explants (where a is callus induction using leaves as explants; b is callus induction using hypocotyls as explants; and c is callus induction using radicles as explants);

[0035] Figure 2 Callus tissue that produces green buds;

[0036] Figure 3 Adventitious buds with different growth potentials (among them, a is an adventitious bud induced by S2 culture medium, with yellow leaves and weak growth; b is an adventitious bud induced by S9 culture medium, with green leaves and good growth). DETAILED DESCRIPTION

[0037] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0038] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The experimental materials, reagents, drugs, etc. used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0039] The Melilotus serrata used in the following examples is Melilotus serrata, and the seeds were purchased from Jiangsu Luzhichang Seed Co., Ltd.

[0040] In the following examples, both MS medium and B5 medium include two major parts: inorganic matter and organic matter, and their specific components are as follows:

[0041] The composition of MS medium is:

[0042] Macroelements: NH 4 NO 3 1650mg / L, KNO 3 1900mg / L, CaCl 2 ·2H 2 O 440mg / L, MgSO 4 7H 2 O370mg / L, KH 2 PO 4170 mg / L;

[0043] Trace elements: KI 0.83 mg / L, H 3 BO 3 6.2 mg / L, MnSO 4 ·4H 2 O 22.3 mg / L, ZnSO 4 ·7H 2 O 8.6 mg / L, Na 2 MoO 4 ·2H 2 O 0.25 mg / L, CuSO 4 ·5H 2 O 0.025 mg / L, CoCl 2 ·6H 2 O 0.025 mg / L;

[0044] Iron salts: FeSO 4 ·7H 2 O 27.8 mg / L, Na 2 -EDTA·2H 2 O 37.3 mg / L;

[0045] Organic substances: inositol 100 mg / L, niacin 0.5 mg / L, pyridoxine hydrochloride (vitamin B6) 0.5 mg / L, thiamine hydrochloride (vitamin B1) 0.1 mg / L, glycine 2.0 mg / L.

[0046] The composition of B5 medium is:

[0047] Inorganic components: sodium dihydrogen phosphate (NaH 2 PO 4 ·H 2 O): 150 mg / L, anhydrous calcium chloride (CaCl 2 ) : 113.24 mg / L, potassium nitrate (KNO 3 ) : 2500 mg / L, ammonium sulfate (NH 4 ) 2 SO 4 : 134 mg / L, anhydrous magnesium sulfate (MgSO 4 ·7H 2 O): 122.09 mg / L, disodium ethylenediaminetetraacetate (Na 2 -EDTA): 37.3 mg / L, ferrous sulfate (FeSO 4 ·7H 2 O): 27.8 mg / L, manganese sulfate (MnSO 4 ·2H 2 O): 10.0 mg / L, zinc sulfate (ZnSO4 7H 2 O): 2.0 mg / L, boric acid (H 3 BO 3 ): 3.0mg / L, sodium molybdate (Na 2 MoSO 4 ·2H 2 O): 0.25 mg / L, copper sulfate (CuSO 4 ·5H 2 O): 0.025 mg / L, cobalt chloride (CoCl 2 6H 2 O): 0.025 mg / L, potassium iodide (KI): 0.75 mg / L;

[0048] Organic ingredients: Vitamin B1 (thiamine hydrochloride): 10.0 mg / L, Vitamin B6 (pyridoxine hydrochloride):

[0049] 1.0mg / L, niacin: 1.0mg / L, inositol: 100.0mg / L.

[0050] Example 1

[0051] A culture medium for Melilotus chinensis tissue culture comprises a callus induction medium, a redifferentiation sprout induction medium and a rooting medium.

[0052] Wherein, the callus induction medium consists of:

[0053] MS medium + 0.5 mg / L 2,4-D + 0.25 mg / L 6-BA + 0.25 mg / L NAA + 30 g / L sucrose + 8 g / L agar, pH 5.8-6.0.

[0054] The redifferentiation sprouting induction medium is composed of: MS medium (without organic matter)+B5 medium organic matter+1mg / L6-BA+0.2mg / L IAA+10mL / L pure coconut water+30g / L sucrose+8g / L agar, and the pH is 5.8-6.0.

[0055] The rooting medium is composed of any one of the following culture media:

[0056] Rooting medium 1: 1 / 2MS medium + 0.2 mg / L IBA + 15 g / L sucrose + 5 g / L agar, pH 5.8-6.0.

[0057] Rooting medium 2: 1 / 2MS medium + 0.2 mg / L NAA + 15 g / L sucrose + 5 g / L agar, pH 5.8-6.0.

[0058] Example 2

[0059] A tissue culture method of Melilotus officinalis, comprising the following steps:

[0060] (1) Cut the hypocotyl and radicle of the sterile seedlings of Melilotus officinalis, make cuts on the leaf surface, and transfer them to callus induction medium to induce callus. The induction conditions are: 8 / 16h dark / light time, 22±2℃ temperature, 50-70% air humidity, 1000-2000lx light intensity, and culture for 14-28 days.

[0061] The callus induction culture medium consists of: MS culture medium + 0.5 mg / L 2,4-D + 0.25 mg / L 6-BA + 0.25 mg / L NAA + 30 g / L sucrose + 8 g / L agar, with a pH of 5.8-6.0.

[0062] (2) Select callus tissues with light yellow color, clumping structure and large callus blocks and inoculate them on redifferentiation bud induction medium to induce the formation of callus differentiation buds. The induction conditions are: 8 / 16h dark / light time, 24±2℃ temperature, air humidity 50-70%, light intensity 2000-3000lx, and culture for 14-28 days.

[0063] The redifferentiation sprouting induction medium is composed of: MS medium (without organic matter)+B5 medium organic matter+1mg / L6-BA+0.2mg / L IAA+10mL / L pure coconut water+30g / L sucrose+8g / L agar, and the pH is 5.8-6.0.

[0064] (3) The buds induced in step (2) are placed in MS solid medium (MS + 30 g / L sucrose + 8 g / L agar, pH 5.8-6.0) without any hormones for cultivation. When they grow to 3-5 cm, they are transferred to rooting medium for rooting induction. The induction conditions are: 8 / 16 h dark / light time, 24±2° C. temperature, 50-70% air humidity, 2000-3000 lx light intensity, and culture for 14-28 days.

[0065] The rooting medium is composed of any one of the following culture media:

[0066] Rooting medium 1: 1 / 2MS medium + 0.2 mg / L IBA + 15 g / L sucrose + 5 g / L agar, pH 5.8-6.0.

[0067] Rooting medium 2: 1 / 2MS medium + 0.2 mg / L NAA + 15 g / L sucrose + 5 g / L agar, pH 5.8-6.0.

[0068] The described Melilotus chinensis sterile seedlings can be obtained by existing methods, or by the following method:

[0069] Select the seeds of Melilotus officinalis with full grains and excellent quality, wash them twice with distilled water and put them in a small beaker for later use. Aseptically operate on a clean bench, first puncture with 75% alcohol for 30 seconds, wash with sterile water 3-5 times, and then disinfect the seeds with 25% sodium hypochlorite solution, and shake for 15 minutes. After shaking, wash with sterile water 3-5 times, dry the surface of the seeds with filter paper, and then inoculate them on a solidified medium with only agar (8g / L agar, pH 5.8-6.0), and place them in an incubator for culture at a temperature of 25±2℃, 16h light / 8h dark.

[0070] Effects of different callus induction media on callus induction of Melilotus officinalis

[0071] Take the hypocotyl and radicle of the sterile seedlings of Melilotus officinalis, cut the leaf surface, and use callus induction medium with different hormone combinations to induce callus tissue on the hypocotyl, radicle, and leaf of Melilotus officinalis. The composition of the culture medium is: MS medium + different hormone combinations + 30g / L sucrose + 8g / L agar. The specific hormone combination is shown in Table 1; the induction conditions are: 8 / 16h dark / light time, 22±2℃ temperature, air humidity 50-70%, light intensity 1000-2000lx, and culture for 14-28 days. Observe and record every 7 days, and compare and analyze the effects of different callus induction medium on callus induction. The callus induction rate is calculated using the following formula:

[0072] Callus induction rate (%) = number of callus explants induced / number of inoculated explants × 100

[0073] Table 1 Callus induction medium of Melilotus officinalis with different hormone combinations

[0074]

[0075]

[0076] The results of callus induction using hypocotyls as explants on different callus induction media are shown in Table 2.

[0077] Table 2 Dedifferentiation and induction of callus from hypocotyls of Melilotus officinalis

[0078]

[0079] As shown in Table 2, among the 9 culture media, F4 had the highest callus induction rate, reaching 89%, with light yellow granular callus. The culture medium formula was MS medium + 0.5mg / L 2,4-D + 0.25mg / L 6-BA + 0.25mg / L NAA + 30g / L sucrose + 8g / L agar, with a pH of 5.8-6.0. It can be seen that different hormones and their concentrations have a significant effect on callus induction. In the culture medium without any hormones, there was no dedifferentiation of the explants. The culture medium with different contents of 2,4-D, NAA, 6-BA, etc. can induce callus to different degrees, with an induction rate of 34%-89%. The combination of only adding NAA and 2,4-D has a certain induction effect on the callus of Melilotus officinalis, but it is seriously waterlogged; adding 6-BA significantly improves the waterlogging phenomenon of the callus. With the increase of 2,4-D content, callus induction first increased and then decreased, indicating that relatively high concentrations of 2,4-D are not conducive to callus induction. When 2,4-D is used together with cytokinin 6-BA, its effect will be enhanced.

[0080] The results of callus induction using leaves as explants on different callus induction media are shown in Table 3.

[0081] Table 3 Dedifferentiation and induction of callus from leaves of Melilotus officinalis

[0082]

[0083] As shown in Table 3, compared with the hypocotyl, the highest callus induction rate of leaf callus was 65% (F7 combination, see Table 3), but most of the callus was water-soaked and non-embryonic callus that was not easy to differentiate into buds.

[0084] When callus was induced on different callus induction media using radicle as explant, only F7 combination could induce very small part of light green callus, with an induction rate of only 8%, and the other combinations could hardly induce callus.

[0085] The callus induction of different explants is shown in Figure 1 .

[0086] In conclusion, hypocotyl is the best explant for inducing callus of Melilotifolia.

[0087] Effects of different redifferentiation bud induction media on bud induction of callus of Melilotifolia

[0088] Select the light yellow, clumping and larger callus tissues of Melilotus officinalis induced by hypocotyl dedifferentiation and inoculate them on different redifferentiation bud induction mediums for bud induction. The differentiation bud formation rate is calculated using the following formula:

[0089] Differentiation bud formation rate (%) = number of differentiation buds formed / number of inoculated callus tissues × 100

[0090] The compositions of different redifferentiation sprouting induction medium are shown in Table 4, and also contain 30 g / L sucrose + 8 g / L agar.

[0091] Table 4 Different redifferentiation budding induction medium

[0092]

[0093]

[0094] Note: “-”: no B5 organic matter is added; “+”: B5 organic matter replaces MS organic matter

[0095] The results of induction of redifferentiation and budding of Melilotus officinalis callus by different redifferentiation and budding induction medium are shown in Table 5.

[0096] Table 5 Results of callus redifferentiation and bud induction of Melilotus officinalis

[0097]

[0098] Callus tissue will basically form buds on the redifferentiation bud induction medium ( Figure 2 ), the formation rate of differentiated buds was low at 17% under the induction condition of single hormone 6-BA (1mg / L). In the medium with 6-BA and IAA hormone combination, the differentiation rate of adventitious buds increased, especially under the condition of 6-BA 1mg / L+IAA 0.2mg / L, a large number of buds were formed on the surface of callus tissue, and the differentiated adventitious buds grew well, but the budding rate was still low at 56%. In addition, there were more buds at high concentration of 6-BA (>2mg / L), but the induction rate of adventitious buds was reduced, indicating that high concentration of 6-BA would inhibit the redifferentiation of adventitious buds of Melilotus officinalis.

[0099] The time of redifferentiation of adventitious buds in the medium with only coconut water added is earlier than the time of adventitious buds in the medium with only B5 medium organic matter added; the growth of adventitious buds in the medium with only B5 medium organic matter added is better than that in the medium with only coconut water added; the medium with coconut water and B5 medium organic matter added has richer nutrients, buds are formed in 5-7 days, and has a higher adventitious bud differentiation efficiency (85%), and the adventitious buds grow best. Therefore, adding coconut water and B5 medium organic matter to the redifferentiation bud induction medium of Melilotus officinalis will greatly improve the efficiency and quality of adventitious bud induction. The different growth conditions of adventitious buds can be seen in Figure 3 .

[0100] Effects of different rooting media on the rooting of Melilotus officinalis

[0101] The induced Melilotus officinalis buds were placed in a culture medium without any hormones for cultivation. When they grew to 4-5 cm, they were transferred to different rooting culture media to induce rooting. The rooting rate was calculated after 30 days.

[0102] The composition of the rooting medium is as follows:

[0103] Rooting medium 1: 1 / 2MS medium + 0.2 mg / L IBA + 15 g / L sucrose + 5 g / L agar, pH 5.8-6.0.

[0104] Rooting medium 2: 1 / 2MS medium + 0.2 mg / L NAA + 15 g / L sucrose + 5 g / L agar, pH 5.8-6.0.

[0105] The rooting rate was calculated using the following formula:

[0106] Rooting rate (%) = number of rooted seedlings / number of seedlings inoculated × 100

[0107] The results of the study found that the differentiated seedlings of Melilotus officinalis can take root in both rooting media. The number and thickness of roots in the rooting medium with IBA added are better than those in the medium with NAA added. The rooting speed is fast, and roots can be formed in about 10 days. The rooting rate is high, up to 75%. The medium with NAA added has a rooting rate of 67%, and the adventitious roots are thinner. After the seedlings with strong root systems are acclimated, they are transplanted into the soil (soil: vermiculite = 2:1). According to statistics, the transplant survival rate can reach 85%. When transplanting, the root culture medium should be cleaned to prevent it from affecting the transplanting results.

[0108] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A medium for tissue culture of Melilotus scabra, characterized in that: The culture medium comprises a callus induction medium, a redifferentiation sprout induction medium, and a rooting medium; The callus induction medium comprises: MS medium + 0.5 mg / L 2,4-D + 0.25 mg / L 6-BA + 0.25 mg / L NAA + 30 g / L sucrose + 8 g / L agar, with a pH of 5.8-6.0; The redifferentiation sprout induction medium comprises: MS medium without organic matter + B5 medium organic matter + 1 mg / L 6-BA + 0.2 mg / L IAA + 10 mL / L pure coconut water + 30 g / L sucrose + 8 g / L agar, with a pH of 5.8-6.0; The rooting medium is composed of any one of the following culture media: Rooting medium 1: 1 / 2MS medium + 0.2mg / L IBA + 15g / L sucrose + 5g / L agar, pH 5.8-6.0; Rooting medium 2: 1 / 2MS medium + 0.2 mg / L NAA + 15 g / L sucrose + 5 g / L agar, pH 5.8-6.

0.

2. Use of the culture medium for tissue culture of Melilotus luteus according to claim 1 in tissue culture of Melilotus luteus.

3. A tissue culture method of Melilotus scabra, characterized in that: Here are the steps: (1) taking an explant of Oleander and inoculating it into a callus induction medium to induce callus; (2) The light yellow callus with a clumping structure and larger callus blocks induced in step (1) are inoculated on a redifferentiation bud induction medium to induce the formation of callus differentiation buds; (3) The buds induced in step (2) are placed in MS solid culture medium without any hormones for cultivation, and when they grow to 3-5 cm, they are transferred to rooting medium to induce rooting; The callus induction medium comprises: MS medium + 0.5 mg / L 2,4-D + 0.25 mg / L 6-BA + 0.25 mg / L NAA + 30 g / L sucrose + 8 g / L agar, pH 5.8-6.0; The redifferentiation sprout induction medium comprises: MS medium without organic matter + B5 medium organic matter + 1 mg / L 6-BA + 0.2 mg / L IAA + 10 mL / L pure coconut water + 30 g / L sucrose + 8 g / L agar, with a pH of 5.8-6.0; The rooting medium is composed of any one of the following culture media: Rooting medium 1: 1 / 2MS medium + 0.2mg / L IBA + 15g / L sucrose + 5g / L agar, pH 5.8-6.0; Rooting medium 2: 1 / 2MS medium + 0.2mg / L NAA + 15g / L sucrose + 5g / L agar, pH 5.8-6.0; The alismatis explant is the hypocotyl of the aseptic seedling of alismatis alismatis.

4. The tissue culture method of Melilotus erythrorhizome according to claim 3, characterized in that: The callus induction conditions in step (1) are: dark / light time 8 / 16h, temperature 22±2°C, air humidity 50-70%, light intensity 1000-2000lx, and culture for 14-28 days; The conditions for inducing callus differentiation and bud formation in step (2) are: darkness / light time 8 / 16h, temperature 24±2°C, air humidity 50-70%, light intensity 2000-3000lx, and culture for 14-28 days; The rooting induction conditions in step (3) are: darkness / light time 8 / 16h, temperature 24±2°C, air humidity 50-70%, light intensity 2000-3000lx, and cultivation for 14-28 days.

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