A method for rapid propagation of Zhejiang red camellia clones through tissue culture

Abstract

The invention discloses a method for rapid propagation of Zhejiang red camellia clones through tissue culture. Using unlignified young stem segments as explants, after sterilization, primary induction, secondary proliferation, strong bud culture and rooting culture are carried out to establish a technical system for rapid propagation of Zhejiang red camellia clones through tissue culture. Before primary inoculation, the explants are soaked, and a certain concentration of vitamin C and polyvinyl pyrrolidone are added to the primary induction medium, which can effectively inhibit the browning of the explants; combined with hormone regulation and nutrient addition, the primary induction rate is as high as 95.3%, the proliferation coefficient is about 6.8, and the rooting rate is 83.5%, which can achieve efficient and rapid propagation and is not limited by factors such as season, climate and region. The rapid propagation offspring obtained by the system can maintain all the excellent traits of the mother plant, and the strong buds obtained in the process can also be used as spikes for grafting and propagation, thereby providing technical support for the protection and development and utilization of rare germplasm resources and excellent asexual systems of Zhejiang red camellia.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for rapid tissue culture of asexual lines of Camellia zekinensis. Background Art

[0002] Zhejiang Red Camellia Camellia chekiangoleosa Hu) is also known as Zhejiang safflower oil tea, belonging to the Theaceae Camellia genus Camellia Sect. Camellia , is a unique tree species in my country, distributed in Zhejiang, Jiangxi, Fujian and Anhui, mostly grown in hillside mixed woods, valley forests, forest edges or streams. Zhejiang Red Camellia is an evergreen small tree with beautiful tree shape, dark green leaves, early spring flowering, large, bright and dense flowers, and apple-like fruits, bright red or yellow-green in color. The leaves, flowers and fruits are of high ornamental value and suitable for cultivation and viewing in gardens. Zhejiang Red Camellia is also an important woody edible oil plant. Its seed kernel oil is rich in unsaturated fatty acids such as oleic acid and linoleic acid, and is known as "Oriental Olive Oil". It also contains trace components such as vitamin E, squalene, and sterols. It is a functional edible oil with multiple health benefits. At the same time, its seed kernel oil content is as high as about 60%, and the oleic acid content in tea oil is as high as about 85%, which are significantly higher than those of white flower tea species such as ordinary tea, and have important economic value. Most of the Zhejiang Red Camellia resources are distributed in a wild or semi-wild state, and their natural habitats have been severely damaged, close to endangered. For the protection and development of rare germplasm resources and excellent asexual lines of Zhejiang Camellia, it is urgent to establish efficient and stable rapid propagation technology.

[0003] Theaceae plants are usually propagated by sowing, cuttings, grafting and tissue culture. Due to the highly heterozygous genetic background, the offspring of Theaceae plants propagated by sowing are severely differentiated, and it is difficult to maintain the excellent traits of the mother plant. For rare germplasm resources and excellent asexual lines, the propagation coefficient of cuttings and grafting is low. If the number of spikes is limited, large-scale propagation is not possible, and it is difficult to meet the needs of large-scale production. At the same time, sowing, cuttings and grafting propagation are restricted by season and region. Tissue culture is a propagation method widely used in agriculture and forestry, with the characteristics of fast propagation speed and no seasonal and regional restrictions.

[0004] For the tissue culture of Zhejiang red camellia, predecessors used young embryo cotyledons, seed cotyledons and anthers as explants, respectively, to establish a somatic embryo regeneration pathway mediated by callus or directly occurring. Since Camellia plants are highly heterozygous, according to genetic laws, the offspring bred with their reproductive organs will have serious trait separation. Therefore, the above-mentioned reproduction offspring obtained by tissue culture with young embryo cotyledons, seed cotyledons and anthers as explants will produce trait separation, and it is difficult to maintain all the excellent characteristics of the mother plant. Using vegetative organs as explants for tissue culture can obtain offspring with the same heredity as the mother plant, thereby maintaining the excellent characteristics of the mother plant while rapidly propagating. At present, there is no report on the relevant technology of Zhejiang red camellia tissue culture with vegetative organs as explants at home and abroad. The present invention uses young stem segments as explants to establish a tissue culture rapid propagation method, which provides an effective way for the stable rapid propagation of rare germplasm resources and excellent clones of Zhejiang red camellia, and provides technical support for its protection and development and utilization. Summary of the invention

[0005] The purpose of the invention is to provide a method for the rapid propagation of Zhejiang red camellia clones through tissue culture in view of the problem that the prior art lacks a method for the rapid propagation of Zhejiang red camellia tissue culture that can maintain the excellent traits of the mother plant.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for rapid tissue culture of asexual lines of Camellia chinensis, comprising the following steps:

[0008] 1) Explant collection: Take young and tender stem segments that are not lignified as explants;

[0009] 2) Explant sterilization: including immersion and shaking sterilization with 75% alcohol solution; immersion and shaking sterilization with 0.1% mercuric chloride solution;

[0010] 3) Primary induction: Remove the two ends of the sterilized stem segment, cut it into small segments with a single node, soak it in sterile water, dry the surface water, and inoculate it into the primary induction medium according to polarity and culture it for 40 to 60 days;

[0011] 4) Subculture proliferation: After the leaves of the buds induced in the primary generation are appropriately cut off, they are inoculated into the subculture proliferation medium and cultured for 30 to 50 days;

[0012] 5) Strong bud culture: The clustered buds obtained from subculture proliferation culture are divided into single buds and inoculated into strong bud culture medium according to polarity and cultured for 30 to 50 days;

[0013] 6) Rooting culture: Take a strong single bud after strong bud culture, remove the leaves close to the base, and cut off 1-3 mm of the base to form a new wound; immerse the base in a rooting solution containing 3 g / L NAA and 2 g / L IBA for 8-12 minutes, and then inoculate it in a rooting medium and culture it for 40-60 days.

[0014] The culture conditions of steps 3), 4), 5) and 6) are: temperature 23-27°C, light intensity 2500-3000 Lx, and light intensity of 14-16 h per day for alternating light and dark culture.

[0015] 1) Explant collection: After 2 to 3 consecutive days of sunny weather, the leaves of the plants are allowed to dry naturally during the day and then young, non-lignified stem segments are collected as explants.

[0016] 2) Explant sterilization: After the leaves are cut off, the stem segments are transferred to a clean bench after cleaning, immersed in 75% alcohol solution and shaken for sterilization for 25 to 35 seconds, rinsed with sterile water for 2 to 3 times, immersed in 0.1% mercuric chloride solution and shaken for sterilization for 11 to 13 minutes, and rinsed with sterile water for 4 to 5 times.

[0017] 3) Primary induction: remove 3-5 mm from both ends of the sterilized stem segments, cut them into small segments with single nodes, soak them in sterile water for 25-35 min, dry the surface moisture with sterile filter paper, inoculate them in primary induction medium according to polarity and culture them for 40-60 days; the formula of the primary induction medium is: MS + 6-BA 0.8-1.2 mg / L + NAA 0.1-0.3 mg / L + GA30.8-1.2 mg / L + VC 3-5 g / L + PVP 3-5 g / L + sucrose 25-35 g / L + agar 6.5-7.5 g / L; pH value is 5.6-5.8.

[0018] 4) Subculture proliferation: The formula of the subculture proliferation medium is: WPM + 6-BA 2.5-3.5 mg / L + TDZ0.04-0.06 mg / L + IBA 0.4-0.6 mg / L + sucrose 25-35 g / L + agar 6.5-7.5 g / L; pH value is 5.6-5.8.

[0019] 5) Strong bud culture: The formula of the strong bud culture medium is: MS + 6-BA 0.4-0.6 mg / L + NAA 0.04-0.06 mg / L + coconut milk 40-60 mL / L + sucrose 25-35 g / L + agar 6.5-7.5 g / L; pH value is 5.6-5.8.

[0020] 6) Rooting culture: The formula of the rooting medium is: 1 / 2 MS + NAA 0.8-1.2 mg / L + IBA 0.4-0.6 mg / L + coconut milk 40-60 mL / L + sucrose 25-35 g / L + agar 6.5-7.5 g / L; pH value is 5.6-5.8.

[0021] The beneficial technical effect of the present invention is as follows: compared with the prior art, the present invention provides a method for rapid propagation of Zhejiang red camellia clones through tissue culture, using unlignified young stem segments as explants, and establishing a tissue culture rapid propagation technology system through primary induction, secondary proliferation, strong bud culture and rooting culture, forming complete tissue culture offspring plants, and the offspring plants can maintain all the excellent traits of the mother plant. The primary induction rate of the system is as high as 95.3%, the proliferation coefficient reaches about 6.8, the rooting rate reaches 83.5%, and it is not limited by factors such as season, climate and region, and can achieve efficient and rapid propagation throughout the year. The strong buds obtained in the system can also be used as spikes for grafting propagation. The present invention can provide technical support for the protection and development and utilization of rare germplasm resources and excellent asexual systems of Zhejiang red camellia.

[0022] For rare germplasm resources with fewer material sources, reducing the explant contamination rate and mortality rate after primary inoculation is a prerequisite for successfully establishing a tissue culture system. The present invention controls the explant inoculation contamination rate and mortality rate within 10% and 5% respectively by controlling the timing of explant collection and the duration of sterilization, thereby providing a prerequisite guarantee for the establishment of a tissue culture system.

[0023] The stem segments of Camellia sinensis are rich in phenolic substances, which are the key factors causing the browning of explants. The present invention soaks the explants before primary inoculation to reduce the phenolic substances in the incision, and at the same time adds a certain concentration of vitamin C (VC) and polyvinyl pyrrolidone (PVP) to the primary induction medium to inhibit the oxidation of phenolic substances, thereby effectively inhibiting the browning of the explants and improving the success rate of primary induction. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 It is the non-lignified young stem segment explant collected in sunny weather in Example 1.

[0026] Figure 2 After the stem segment explants of Example 2 were inoculated and subjected to primary induction culture, axillary buds began to sprout.

[0027] Figure 3 The axillary buds after the initial induction in Example 2 grew to 4-5 cm.

[0028] Figure 4 This is the axillary bud subculture proliferation of Example 3.

[0029] Figure 5 This is the strong bud culture after proliferation in Example 4.

[0030] Figure 6 It is the rooting culture after the strong bud of Example 5. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with specific embodiments. These embodiments should be understood as merely preferred examples of the present invention, rather than limiting the present invention in any way.

[0032] Example 1: Effect of explant collection method and sterilization treatment on the present invention

[0033] 1) Experimental materials: In April and May, healthy plants of Camellia sinensis over 5 years old were selected, and young stem segments of the current year without lignification were cut as explant materials ( Figure 1 ).

[0034] 2) Experimental methods: Two explant sampling methods: ① Randomly select a certain time period of a day to sample; ② Sample after the surface of the plant leaves naturally dry during the day after 2 to 3 consecutive sunny days. Sterilization treatment uses 75% alcohol immersion and shaking, rinse with sterile water 2 to 3 times, 0.1% mercuric chloride solution or 2% sodium hypochlorite solution immersion and shaking, rinse with sterile water 4 to 5 times. There are five treatments for immersion and shaking: ① 75% alcohol 30 s, 0.1% mercuric chloride solution 8 min; ② 75% alcohol 30 s, 0.1% mercuric chloride solution 12 min; ③ 75% alcohol 30 s, 0.1% mercuric chloride solution 16 min; ④ 75% alcohol 60 s, 0.1% mercuric chloride solution 12 min; ⑤ 75% alcohol 30 s, 2% sodium hypochlorite solution 10 min. After inoculation and cultivation, the number of explant contamination and death was counted, and the explant contamination rate and mortality rate were calculated. The contamination rate (%) = the number of contaminated explants / the total number of inoculated explants × 100, and the explant mortality rate (%) = the number of dead explants / the total number of inoculated explants × 100.

[0035] 3) Experimental results and conclusions:

[0036]

[0037] Comparative analysis of the two sampling methods showed (Table 1) that, compared with ① sampling randomly selected at a certain time of day, ② sampling after the plant leaf surface has dried naturally during the day after 2 to 3 consecutive sunny days can significantly reduce the contamination of explants.

[0038]

[0039] The effects of different sterilization treatments on explant inoculation were observed. It was found that after treatment with ⑤75% alcohol for 30 s and 2% sodium hypochlorite solution for 10 min, the explant mortality rate reached 33.5%. Although the stems of some explants remained green, the buds on them turned black and lost vitality. This may be due to the strong alkalinity of sodium hypochlorite, which caused certain damage to the explant materials. In the combined treatment of 75% alcohol and 0.1% mercuric chloride solution, the explant mortality rate also reached 28.7% after treatment with ④75% alcohol for 60 s and 0.1% mercuric chloride solution for 12 min, indicating that the 75% alcohol treatment time should not be too long. Comparing the three treatments of ①, ② and ③, the contamination rate and mortality rate of the explants after treatment with ②75% alcohol for 30 s and 0.1% mercuric chloride solution for 12 min were both low, at 8.9% and 4.6%, respectively (Table 2).

[0040] Therefore, after 2 to 3 consecutive sunny days, samples are taken after the surface of the plant leaves are naturally dried during the day, and 75% alcohol for 30 seconds and 0.1% mercuric chloride solution for 12 minutes are used for sterilization, which are effective methods for collecting and sterilizing young stem segment explants of Zhejiang red camellia. For rare germplasm resources with fewer material sources, reducing the explant contamination rate and mortality rate after primary inoculation is a prerequisite for successfully establishing a tissue culture system. The present invention controls the explant inoculation contamination rate and mortality rate within 10% and 5% respectively by controlling the timing of explant collection and the duration of sterilization, thereby providing a prerequisite guarantee for the establishment of a tissue culture system.

[0041] Example 2: Effect of the primary induction method on the present invention

[0042] 1) Experimental materials: The plant materials used in the experiment were sterilized stem segments that survived sterilization. The culture medium was prepared using commercially available MS basic medium, hormones (6-BA, NAA and GA3), anti-browning agents (VC and PVP), sucrose and agar.

[0043] 2) Experimental method: MS medium was used as the basic medium, and hormone 6-BA (0.5, 1.0, 1.5 mg / L), NAA (0, 0.2, 0.5 mg / L), GA3 (0, 1.0, 2.0 mg / L), anti-browning agents (4 g / L VC and 4 g / L PVP) or no anti-browning agent was added. 30 g / L sucrose and 7 g / L agar were added to all medium, and the pH was adjusted to 5.8 and sterilized at 121 ℃ for 20 min for use. After sterilization, the two ends of the stem segment were removed by 3-5 mm, cut into small segments with a single node, soaked in sterile water for 30 min or not, and the surface moisture was absorbed with sterile filter paper. The lower end of the stem segment was inserted into the culture medium according to polarity, and 2-3 stem segments were inoculated in each bottle and placed on the culture rack in the culture room for primary induction culture. The temperature of the culture room is 23℃~27℃, the light intensity at the bottom of the culture layer is 2500~3000 Lx, and the lighting time is 14~16 h per day.

[0044] Observe and record the condition of the stem segments and the budding of the buds on them, and evaluate the effects of explant immersion treatment, anti-browning agents and hormones on primary induction culture. Statistically calculate the primary induction rate, which is (%) = the number of stem segments that sprouted / the total number of inoculated stem segments × 100.

[0045] 3) Experimental results and conclusions:

[0046]

[0047] ① Before inoculation, the explants were soaked in sterile water for 30 min, and anti-browning agents 4 g / L VC and PVP were added to the culture medium. The ends of the explant stem segments and axillary buds were basically free of browning. The axillary buds began to sprout about 12 days after inoculation ( Figure 2 ), after 30 days, 1-2 cm new shoots emerge, and after 60 days, the new shoots grow to 4-5 cm ( Figure 3 ), the induction rate reached 95.3% (Table 3). ② When the explants were immersed for only 30 min, or ③ when only anti-browning agents were added to the culture medium, some axillary buds were delayed in germination or could not germinate due to browning, and the induction rates were 78.2% and 83.6%, respectively. ④ When the explants were not immersed and the anti-browning agents were not added to the culture medium, the induction rate was only 70.1%.

[0048]

[0049] On the medium without any hormones, the buds were uneven, the buds grew slowly, and the induction rate was only 23.0%. On the medium without GA3, the buds were uneven and grew slowly. On the medium without NAA, the buds were thin. Among all hormone combinations, the medium with 1.0 mg / L 6-BA, 0.2 mg / L NAA, and 1.0 mg / L GA3 added, the induction rate was the highest, reaching 95.3%, and the buds were even, the buds were strong, and the leaves were dark green (Table 4, Figure 3 ).

[0050] Therefore, soaking the explants in sterile water for 30 min before inoculation and adding 4 g / L VC and PVP, an anti-browning agent, to the culture medium can effectively inhibit the browning of the explants and improve the success rate of primary induction. Adding 1.0 mg / L 6-BA, 0.2 mg / L NAA and 1.0 mg / L GA3 to MS medium for primary induction of young stem segments of Camellia chinensis not only has a high induction rate, but also good bud growth.

[0051] Example 3: Effect of Subculture Proliferation Culture Method on the Present Invention

[0052] 1) Experimental materials: The plant materials used in the experiment were new shoots obtained after the axillary buds germinated in the primary induction. The culture medium was prepared using commercially available WPM basic medium, hormones (6-BA, TDZ, NAA and IBA), sucrose and agar.

[0053] 2) Experimental methods: WPM medium was used as the basic medium, and different types and concentrations of hormones were added: 6-BA (2.0, 3.0, 4.0 mg / L), TDZ (0, 0.05, 0.1 mg / L), NAA (0, 0.5, 1.0 mg / L), and IBA (0, 0.5, 1.0 mg / L). 30 g / L sucrose and 7 g / L agar were added to all mediums, and the pH was adjusted to 5.8 and sterilized at 121 ℃ for 20 min for use. The leaves of the new shoots induced in the primary generation were appropriately cut off and inoculated into the subculture proliferation medium, and the culture bottles were placed on the culture rack in the culture room for culture. The temperature in the culture room was 23 ℃ to 27 ℃, the light intensity at the bottom of the culture layer was 2500-3000 Lx, and the light time was 14-16 h per day. The proliferation and growth of the shoots were observed and recorded, and the effect of hormones on subculture proliferation culture was evaluated.

[0054] 3) Experimental results and conclusions:

[0055]

[0056] On the culture medium without any hormones added, the buds grew normally, but no proliferation occurred during the culture period. 6-BA can effectively promote the proliferation of buds, but when its concentration reaches 4.0 mg / L, the leaves become curled and deformed, and the new buds are weak. When the concentration is 3.0 mg / L, the proliferation effect is good, and the buds grow healthily. The addition of TDZ can increase the proliferation coefficient, but when the concentration reaches 0.1 mg / L, some new buds turn brown and die in the later stage. The appropriate concentration is 0.05 mg / L. The auxins NAA and IBA promote the proliferation of buds, among which IBA has a better effect, but its concentration should not be too high. When the concentration reaches 1.0 mg / L, it will cause the leaves to turn yellow, and some apical buds will gradually turn brown and die. Therefore, ② adding 3.0 mg / L 6-BA, 0.05 mg / L TDZ and 0.5 mg / L IBA to the culture medium is beneficial to the proliferation of Zhejiang red camellia in vitro buds. After 40 days of culture, the proliferation coefficient can reach 6.8, and the buds grow healthily (Table 5, Figure 4 ).

[0057] Example 4: Influence of the bud cultivation method on the present invention

[0058] 1) Experimental materials: The plant materials used in the experiment were clustered shoots obtained by proliferation culture. The culture medium was prepared using commercially available MS basic medium, hormones (6-BA and NAA), coconut milk, sucrose and agar.

[0059] 2) Experimental method: MS medium was used as the basic medium, and different concentrations of 6-BA (0.2, 0.5, 1.0 mg / L) and NAA (0.02, 0.05, 0.1 mg / L) were added, and 50 mL / L coconut milk or no coconut milk was added. 30 g / L sucrose and 7 g / L agar were added to all medium, and the pH was adjusted to 5.8 and sterilized at 121 ℃ for 20 min for use. The clustered buds obtained by subculture proliferation culture were divided into single buds, inoculated into strong bud culture medium according to polarity, and the culture bottles were placed on the culture rack in the culture room for culture. The temperature in the culture room was 23 ℃~27 ℃, the light intensity at the bottom of the culture layer was 2500~3000 Lx, and the light time was 14~16 h per day. The growth of the buds was observed and recorded, and the effects of hormones and coconut milk on strong bud culture were evaluated.

[0060] 3) Experimental results and conclusions:

[0061]

[0062] In the medium with ① a lower concentration of cytokinin (0.2 mg / L 6-BA) or ⑤ a higher concentration of auxin (0.1 mg / L NAA), the buds grew slowly. In the medium with 0.5-1.0 mg / L 6-BA and 0.02-0.05 mg / L NAA, the buds grew fast. The addition of coconut juice can promote strong buds. In the medium with ② 0.5 mg / L 6-BA, 0.05 mg / L NAA and 50 mL / L coconut juice, not only did the buds grow fast, but also the stems were thick and the leaves were dark green after 40 days of culture (Table 6, Figure 5 ). Therefore, ② is suitable for the in vitro strong bud culture of Zhejiang red camellia.

[0063] Example 5: Effect of Rooting Culture Method on the Present Invention

[0064] 1) Experimental materials: The plant materials used in the experiment are strong single buds obtained after strong bud culture. The culture medium is made using commercially available 1 / 2 MS basic medium, hormones (NAA and IBA), coconut milk, sucrose and agar.

[0065] 2) Experimental method: 1 / 2 MS medium was used as the basic medium, and different concentrations of NAA (0.5, 1.0, 1.5 mg / L) and IBA (0.2, 0.5, 1.0 mg / L) were added, and 50 mL / L coconut milk or no coconut milk was added. 30 g / L sucrose and 7 g / L agar were added to all medium, and the pH was adjusted to 5.8 and sterilized at 121 ℃ for 20 min for use. Take the strong single buds after strong bud culture, remove the leaves close to the base, and cut off 1-3 mm of the base to form a new wound. Immerse the base in the rooting solution containing 3 g / L NAA and 2 g / LIBA for 0-20 min, then inoculate it in the rooting medium, and place the culture bottle on the culture rack in the culture room for culture. The temperature in the culture room is 23 ℃-27 ℃, the light intensity at the bottom of the culture layer is 2500-3000 Lx, and the light time is 14-16 h per day. Observe and record the rooting situation and evaluate the effect of hormones on rooting culture. Rooting rate (%) = number of rooted buds / total number of buds × 100.

[0066] 3) Experimental results and conclusions:

[0067]

[0068] The base of the buds was immersed in a rooting solution containing 3 g / L NAA and 2 g / L IBA for 0-20 min, and then inoculated into a suitable culture medium for culture. The results showed (Table 7) that the immersion treatment could increase the rooting rate, among which the rooting rate was the highest when immersed for 10 min, reaching 83.5%.

[0069]

[0070] After the strong buds are cultured, they are soaked in rooting solution and inoculated into rooting medium. After 15 to 25 days of culture, the base swells and the roots are formed after 20 to 30 days of culture. Figure 6 ). Comparative analysis of rooting in different culture media (Table 8) showed that the highest rooting rate was 83.5% in the culture medium with 1.0 mg / L NAA and 0.5 mg / L IBA added (9). Adding 50 mL / L coconut juice promoted the growth of seedlings, making their leaves dark green and their roots thick and strong.

[0071] The embodiments described above can be further combined or replaced, and the embodiments are only descriptions of preferred embodiments of the present invention, and do not limit the concept and scope of the present invention. Without departing from the design concept of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention belong to the protection scope of the present invention. The protection scope of the present invention is given by the attached claims and any equivalent technical solutions thereof.

Claims

1. A method for rapid tissue culture of asexual plants of Camellia sinensis, characterized in that: include: 1) Explant collection: After 2 to 3 consecutive days of sunny weather, the leaves of the plants were dried naturally during the day and young stem segments that were not lignified were taken as explants; 2) Explant sterilization: After the leaves are cut off, the stem segments are cleaned and transferred to a clean bench. They are then immersed in 75% alcohol solution and shaken for sterilization for 25 to 35 seconds, rinsed with sterile water for 2 to 3 times, immersed in 0.1% mercuric chloride solution and shaken for sterilization for 11 to 13 minutes, and rinsed with sterile water for 4 to 5 times. 3) Primary induction: remove 3-5 mm from both ends of the sterilized stem segments, cut into small segments with single nodes, soak in sterile water for 25-35 min, dry the surface moisture with sterile filter paper, inoculate in primary induction medium according to polarity and culture for 40-60 days; the formula of the primary induction medium is: MS + 6-BA 0.8-1.2 mg / L + NAA 0.1-0.3 mg / L + GA3 0.8-1.2 mg / L + VC 3-5 g / L + PVP 3-5 g / L + sucrose 25-35 g / L + agar 6.5-7.5 g / L; pH value is 5.6-5.8; 4) Subculture proliferation: After the leaves of the buds induced in the primary generation are appropriately cut off, they are inoculated in the subculture proliferation medium and cultured for 30 to 50 days; the formula of the subculture proliferation medium is: WPM + 6-BA 2.5 to 3.5 mg / L + TDZ 0.04 to 0.06 mg / L + IBA 0.4 to 0.6 mg / L + sucrose 25 to 35 g / L + agar 6.5 to 7.5 g / L; pH value is 5.6 to 5.8; 5) Strong bud culture: The clustered buds obtained by subculture proliferation culture are divided into single buds, and inoculated into strong bud culture medium according to polarity and cultured for 30 to 50 days; the formula of the strong bud culture medium is: MS + 6-BA 0.4 to 0.6 mg / L + NAA 0.04 to 0.06 mg / L + coconut milk 40 to 60 mL / L + sucrose 25 to 35 g / L + agar 6.5 to 7.5 g / L; pH value is 5.6 to 5.8; 6) Rooting culture: Take a strong single bud after strong bud culture, remove the leaves close to the base, and cut off 1 to 3 mm of the base to form a new wound; immerse the base in a rooting solution containing 3 g / L NAA and 2 g / L IBA for 8 to 12 minutes, and then inoculate it in a rooting medium and culture it for 40 to 60 days; the formula of the rooting medium is: 1 / 2 MS + NAA 0.8 to 1.2 mg / L + IBA 0.4 to 0.6 mg / L + coconut water 40 to 60 mL / L + sucrose 25 to 35 g / L + agar 6.5 to 7.5 g / L; pH value is 5.6 to 5.8; The culture conditions of steps 3), 4), 5) and 6) are: temperature 23-27°C, light intensity 2500-3000 Lx, and light intensity of 14-16 h per day for alternating light and dark culture.

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