A tissue culture method for goodyera repanda

Abstract

This invention discloses a method for tissue culture of *Anoectochilus pingbianense*, comprising: selecting wild *Anoectochilus pingbianense*, removing leaves and roots, rinsing, cutting stem segments, soaking in alcohol and rinsing with sterile water, soaking in sodium hypochlorite and rinsing with sterile water again to obtain treated stem segments; inoculating the treated stem segments into an induction medium for culture; inoculating the obtained stem segments into a rooting medium for culture; and inoculating the obtained stem segments into a tissue culture seedling medium for culture to obtain tissue culture seedlings with complete roots, stems and leaves. This invention solves the problems of severe browning and long propagation cycle in existing *Anoectochilus pingbianense* tissue culture, which cannot meet production needs, greatly reduces possible contamination during tissue culture, reduces the browning rate of explants, and shortens the growth cycle of tissue culture seedlings.

Description

Technical Field

[0001] This invention relates to a method for tissue culture of traditional Chinese medicinal materials, specifically a method for tissue culture of *Anoectochilus pingbianense*. Background Technology

[0002] Anoectochilus pingbianensis is a plant belonging to the genus Anoectochilus in the Orchidaceae family. It is produced in Pingbian, Yunnan Province and is a national second-class protected plant. It is known as the "King of Medicines" and the whole plant is used as medicine. It is a traditional and precious Chinese medicinal material. Existing literature 1 (Shen Tingming, Liu Zhiyuan, et al. Development and utilization value and industrialization development ideas of Anoectochilus pingbianensis[J]. Strait Pharmaceutical Journal, 2016, 8(28):8-9) discloses that Anoectochilus pingbianensis contains alkaloids, trace elements, amino acids, sugars and flavonoids, and has the effects of lowering blood sugar, nourishing yin and reducing fire, anti-inflammatory and analgesic, anti-aging, promoting body fluid and beautifying the skin, and prolonging life.

[0003] Reference 2 (Chen Qiangwei. Study on the in vitro and in vivo antioxidant effects and mechanism of artificially cultivated Anoectochilus roxburghii ethanol extract [C] Master's thesis of Guangzhou Pharmaceutical University, 2018) discloses that the seeds of Anoectochilus roxburghii from Pingbian are small and powdery, with incomplete embryo development and low natural reproduction rate; in addition, the natural environment has been severely damaged, and the growth is extremely slow; and insects and birds like to eat it, so natural resources are scarce and the market price is high, reaching 3,000-5,000 yuan / kg, which leads to indiscriminate harvesting by humans. Wild resources are now on the verge of depletion and can no longer meet the needs of clinical medicine and other industries.

[0004] Reference 3 (Zhou Rui, Kong Qiong, et al. Research on rapid propagation technology of wild Anoectochilus roxburghii in Pingbian through tissue culture [J]. Yunnan Agricultural Science and Technology, 2014, 4: 7-10) discloses that the rapid propagation of Anoectochilus roxburghii in Pingbian is mainly carried out through the mass propagation of protocorms and clustered buds [3]. However, the conventional tissue culture method of Anoectochilus roxburghii not only has a high culture contamination rate and severe browning of explants, but also has a long propagation cycle that cannot meet the production needs. Therefore, it is urgent to explore and improve the conditions for tissue culture of Anoectochilus roxburghii in Pingbian. Summary of the Invention

[0005] The purpose of this invention is to provide a method for tissue culture of *Anoectochilus pingbianensis*, which solves the problems of high contamination rate and severe browning in existing tissue culture methods, as well as long propagation cycles that cannot meet production needs. This method greatly reduces potential contamination during tissue culture, lowers the browning rate of explants, and shortens the growth cycle of tissue culture seedlings.

[0006] To achieve the above objectives, the present invention provides a method for tissue culture of *Anoectochilus pingbianense*, the method comprising:

[0007] (1) Stem segment treatment

[0008] Select wild *Anoectochilus pingbianensis*, remove leaves and roots, rinse, cut stem segments of *Anoectochilus pingbianensis*, soak in alcohol and rinse with sterile water, soak in sodium hypochlorite and rinse with sterile water to obtain processed stem segments.

[0009] (2) Adventitious bud induction

[0010] The treated stem segments were inoculated into an induction medium for culture.

[0011] The induction medium consists of MS basal medium, 0.25 mg / L to 1 mg / L of 6-BA, 0.5 mg / L to 2 mg / L of 2,4-D, 0.75 mg / L to 1.5 mg / L of NAA, 10 g / L to 30 g / L of sucrose, 0.2 g / L to 0.8 g / L of activated charcoal powder, and 1 g / L to 2 g / L of plant gel.

[0012] The cultivation was carried out under a light intensity of 40 μmol / m². 2 / s~120μmol / m 2 The experiment was conducted under red and blue light with a relative intensity of (5-7):(3-5) / s;

[0013] (3) Rooting induction

[0014] The stem segments obtained in step (2) were inoculated onto a rooting medium for culture.

[0015] The rooting medium consists of 1 / 2 to 2 MS basal medium, 0.2 mg / L to 0.4 mg / L 6-BA, 0.05 mg / L to 0.15 mg / L IBA, 10 g / L to 40 g / L sucrose and 1.5 g / L plant gel.

[0016] The cultivation was carried out under a light intensity of 40 μmol / m². 2 / s~120μmol / m 2 The experiment was conducted under red and blue light with a relative intensity of (3-5):(5-7) / s;

[0017] (4) Tissue culture seedling culture

[0018] The stem segments obtained in step (3) are inoculated onto tissue culture medium and cultured to obtain tissue culture seedlings with complete roots, stems and leaves;

[0019] The tissue culture medium consists of 1 / 2 to 1 MS basal medium, 3.0 mg / L to 5.0 mg / L 6-BA, 0.3 mg / L to 0.5 mg / L NAA, 10 g / L to 30 g / L sucrose and 1.5 g / L plant gel.

[0020] The cultivation was carried out under a light intensity of 40 μmol / m².2 / s~120μmol / m 2 The experiment was conducted under red and blue light with a relative intensity of (1-7):(0-5) / s.

[0021] Preferably, in step (1), the concentration of sodium hypochlorite is 0.1 g / 100 mL, the stem segment is a mature stem segment, and the length of the stem segment is 0.5 cm.

[0022] Preferably, in step (2), the induction medium consists of MS basal medium, 0.75 mg / L 6-BA, 0.5 mg / L 2,4-D, 1.25 mg / L NAA, 10 g / L sucrose, 0.4 g / L activated carbon powder and 1.5 g / L plant gel, and the pH of the induction medium is 5.6 to 5.8.

[0023] Preferably, in step (2), the culture temperature is 22℃~26℃, the humidity is 70%, the daily light exposure time is 12h, and the culture time is 15~20 days; the light intensity of both red and blue light is 120μmol / m 2 / s, relative intensity is 5:5.

[0024] Preferably, in step (3), the rooting medium consists of 1 / 2 MS basal medium, 0.3 mg / L 6-BA, 0.1 mg / L IBA, 30 g / L sucrose and 1.5 g / L plant gel, and the pH of the rooting medium is 5.6 to 5.8.

[0025] Preferably, in step (3), the culture temperature is 22℃~26℃, the humidity is 70%, the daily light exposure time is 12h, and the culture time is 15~20 days.

[0026] Preferably, in step (3), the illumination intensity of both the red and blue light is 80 μmol / m². 2 / s, relative intensity is 3:7.

[0027] Preferably, in step (4), the tissue culture medium consists of 1 / 2 MS basal medium, 4.0 mg / L 6-BA, 0.4 mg / L NAA, 30 g / L sucrose and 1.5 g / L plant gel, and the pH of the tissue culture medium is 5.6 to 5.8.

[0028] Preferably, in step (4), the culture temperature is 22℃~26℃, the humidity is 70%, the daily light exposure time is 12h, and the culture time is 4~5 months.

[0029] Preferably, in step (4), the illumination intensity of both the red and blue light is 120 μmol / m². 2 / s, relative intensity is 5:5.

[0030] The present invention provides a method for tissue culture of *Anoectochilus pingbianense*, which solves the problems of severe browning and long propagation cycle in existing *Anoectochilus pingbianense* tissue cultures, thus failing to meet production needs. This method has the following advantages:

[0031] This invention, through extensive experimental research, has yielded the optimal selection of explant materials, culture media, and culture conditions, which greatly reduces potential contamination during tissue culture, lowers the browning rate of explants, and shortens the growth cycle of tissue culture seedlings.

[0032] The adventitious bud induction medium was based on MS medium, supplemented with 1.5 g / L plant gel, 10 g / L sucrose, and plant growth hormones of 0.75 mg / L 6-BA, 0.5 mg / L 2,4-D, and 1.25 mg / L NAA, and 0.4 g / L activated charcoal. The study found that different amounts of plant gel significantly affected plant growth. A plant gel concentration of 1.5 g / L was most beneficial for tissue culture of *Anoectochilus pingbianense*. A sucrose concentration of 10 g / L was optimal for inducing adventitious buds in *Anoectochilus pingbianense*. 0.75 mg / L 6-BA, 0.5 mg / L 2,4-D, and 1.25 mg / L NAA were most beneficial for adventitious bud growth. An activated charcoal concentration of 0.4 g / L was optimal for inhibiting browning of *Anoectochilus pingbianense* stem segments. During cultivation on the induction medium, a red-to-blue light ratio of 5:5 and a light intensity of 120 μmol / m² were used. 2 / s conditions significantly accelerated the germination of adventitious buds in *Anoectochilus roxburghii*.

[0033] The rooting induction medium was 1 / 2 MS medium supplemented with 1.5 g / L plant gel, 30 g / L sucrose, and plant growth hormones of 0.3 mg / L 6-BA and 0.1 mg / L IBA. The 1 / 2 MS basal medium of this invention is most suitable for the adventitious root growth of *Anoectochilus pingbianensis*, with a sucrose concentration of 30 g / L optimal for inducing rooting, and the addition of 0.3 mg / L 6-BA and 0.1 mg / L IBA being most beneficial for rooting. A relative intensity ratio of red to blue light of 3:7 and a light intensity of 80 μmol / m² were used. 2 / s is the most suitable for promoting rooting of *Anoectochilus roxburghii*.

[0034] The tissue culture seedling medium consisted of 1 / 2 MS basal medium supplemented with 1.5 g / L plant gel, 30 g / L sucrose, and plant growth hormones of 4.0 mg / L 6-BA and 0.4 mg / L NAA. The combination of a 5:5 ratio of red to blue light intensity and a light intensity of 120 μmol / m² was used. 2 The cultivation conditions of / s are most suitable for the growth of tissue culture seedlings of Anoectochilus roxburghii from Pingbian. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] The sources of the culture media involved in the following examples are as follows:

[0037] MS basal medium (excluding agar and sugar): Purchased from Hangzhou Best Biotechnology Co., Ltd., China. Specification: 250g, Product No.: BS1004, Item No.: 1301.

[0038] Example 1

[0039] A method for tissue culture of *Anoectochilus roxburghii*, the method comprising:

[0040] (1) Stem segment treatment

[0041] Select mature, disease-free wild *Anoectochilus roxburghii* (a type of orchid), remove leaves and roots, rinse thoroughly with clean water, then rinse 5-8 times with deionized water. Place the plants on a clean bench and cut 0.5cm mature stem segments and 0.5cm young stem segments from the *Anoectochilus roxburghii*. Soak each segment in 75% alcohol for 30 seconds, then rinse 5-8 times with sterile water. Soak the stem segments in 0.1g / 100ml sodium hypochlorite solution for 8-10 minutes, and finally rinse 5-8 times with sterile water to obtain the processed stem segments.

[0042] (2) Adventitious bud induction

[0043] The treated stem segments were inoculated into an induction medium sterilized by high temperature and high pressure (121℃, 0.1MPa for 20 min) for culture. The induction medium consisted of MS basal medium.

[0044] The culture medium consisted of 0.75 mg / L 6-BA, 0.5 mg / L 2,4-D, 1.25 mg / L NAA, 10 g / L sucrose, 0.4 g / L activated charcoal powder, and 1.5 g / L plant gel. The pH was 5.6–5.8. The culture conditions were: temperature 24 ± 2℃, humidity 70%, red to blue light intensity ratio 5:5, daily light duration 12 h, and light intensity 120 μmol / m². 2 / s, the culture time is 15-20 days;

[0045] (3) Rooting induction

[0046] The stem segments obtained in step (2) were inoculated onto a rooting medium sterilized by high temperature and high pressure (sterilization at 121℃ and 0.1MPa for 20 min) and cultured. The rooting medium consisted of 1 / 2 MS basal medium.

[0047] The formula consisted of 0.3 mg / L 6-BA, 0.1 mg / L IBA, 30 g / L sucrose, and 1.5 g / L plant gel, with a pH of 5.6–5.8. The culture conditions were: temperature 24 ± 2℃, humidity 70%, relative intensity ratio of red to blue light 3:7, daily light duration 12 h, and light intensity 80 μmol / m². 2 / s, with a culture time of 15-20 days;

[0048] (4) Tissue culture seedling culture

[0049] The stem segments obtained in step (3) were inoculated onto tissue culture medium that had been sterilized by high temperature and high pressure (121℃, 0.1MPa for 20 min). The stem segments were laid flat during inoculation. After 4 to 5 months of culture, tissue culture seedlings with complete roots, stems and leaves were obtained. The tissue culture medium consisted of 1 / 2 MS basal medium.

[0050] The formula consisted of 4.0 mg / L 6-BA, 0.4 mg / L NAA, 30 g / L sucrose, and 1.5 g / L plant gel, with a pH of 5.6–5.8. The culture conditions were: temperature 24 ± 2℃, humidity 70%, red to blue light intensity ratio of 5:5, daily light duration 12 h, and light intensity of 120 μmol / m². 2 / s, the culture time is 4 to 5 months.

[0051] Experiment Example 1 investigated the optimal concentration of activated carbon powder to inhibit browning of stem segments of *Anoectochilus roxburghii*.

[0052] Mature and young stem segments treated in Example 1 were inoculated into commonly used tissue culture medium for *Anoectochilus roxburghii* (MS + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose), with different concentrations of activated charcoal powder added: 0.2 g / L, 0.4 g / L, 0.6 g / L, or 0.8 g / L. Eight identical stem segments (four mature and four young) were inoculated into each bottle of medium of equal quality, with each treatment replicated 10 times. After 20 days, the medium contamination rate, stem browning rate, and adventitious bud germination rate were calculated. Medium contamination rate (%) = number of contaminated bottles per treatment / total number of inoculated bottles per treatment × 100%; browning rate (%) = number of browned stem segments per treatment / total number of inoculated stem segments per treatment × 100%; adventitious bud germination rate (%) = number of stem segments with adventitious bud germination per treatment / total number of inoculated stem segments per treatment × 100%. The results are detailed in Table 1.

[0053] Table 1: Statistics on contamination rate, browning rate and adventitious bud germination rate of *Anoectochilus pingbianensis* tissue culture with different concentrations of activated carbon powder and different stem segments.

[0054]

[0055] Table 1 shows that the contamination and browning rates of mature stem segments were significantly lower than those of young stem segments, while the germination rate of adventitious buds in mature stem segments was significantly higher than that in young stem segments (the germination of adventitious buds in contaminated young stem segments was not included). Furthermore, an activated carbon powder concentration of 0.4 g / L was found to be optimal for inhibiting browning in *Anoectochilus roxburghii* stem segments.

[0056] Example 2: Optimization of Adventitious Bud Induction Culture Medium and Culture Conditions

[0057] 1. Selection of basal culture medium

[0058] Based on the commonly used medium for Anoectochilus roxburghii tissue culture (MS + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 0.4 g / L activated charcoal powder), two additional commonly used mediums were added (the compositions of the two commonly used mediums were 2 MS + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 0.4 g / L activated charcoal powder and 1 / 2 MS + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 0.4 g / L activated charcoal powder, respectively). Six identical mature stem segments were inoculated into each bottle of medium of equal quality, and each treatment was replicated 10 times. The adventitious bud germination rate was counted after 20 days. The results are detailed in Table 2.

[0059] Table 2: Statistics on the germination rate of adventitious buds of *Anoectochilus pingbianense* under different basal culture media

[0060]

[0061] As shown in Table 2, the adventitious bud germination rate of Anoectochilus pingbianensis was optimal with MS basal medium.

[0062] 2. Selection of plant gel concentration

[0063] Based on the commonly used culture medium for *Anoectochilus roxburghii* (MS + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 0.4 g / L activated charcoal powder), 1 g / L, 1.5 g / L, and 2 g / L plant gel were added respectively. Six identical mature stem segments were inoculated into each bottle of medium of equal quality, and each treatment was replicated 10 times. The adventitious bud germination rate was counted after 20 days. The results are detailed in Table 3.

[0064] Table 3: Statistics on the germination rate of adventitious buds of *Anoectochilus pingbianensis* with different plant gel concentrations

[0065]

[0066] As shown in Table 3, the optimal germination rate of adventitious buds for *Anoectochilus roxburghii* was achieved using 1.5 g / L plant gel.

[0067] 3. Selection of sucrose concentration

[0068] Based on the above steps, the commonly used culture medium for *Anoectochilus roxburghii* tissue culture (MS + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 0.4 g / L activated charcoal powder + 1.5 g / L plant gel) was selected. Different concentrations of sucrose (0 g / L, 10 g / L, 20 g / L, and 30 g / L) were added. Six identical mature stem segments were inoculated into each bottle of medium of equal mass. Each treatment was replicated 10 times. The adventitious bud germination rate was calculated after 20 days. The results are detailed in Table 4.

[0069] Table 4: Statistics on the bud germination rate of *Anoectochilus roxburghii* var. *pingbianensis* under different sucrose concentrations

[0070]

[0071] As shown in Table 4, the optimal germination rate of adventitious buds in *Anoectochilus roxburghii* was achieved with a sucrose concentration of 10 g / L.

[0072] 4. Optimization and screening of plant hormone concentrations

[0073] An orthogonal design was used for the experiment (see Table 5). Six identical mature stem segments were inoculated into each bottle of culture medium of equal quality, with each treatment replicated 10 times. Adventitious bud germination was observed 20 days after inoculation. The culture medium consisted of MS medium + 1.5 g / L plant gel + 10 g / L sucrose + 0.4 g / L activated charcoal powder, with a pH of 5.6–5.8. The results are detailed in Tables 5 and 6.

[0074] Table 5: Orthogonal experimental design for inducing plant hormones from adventitious buds of *Anoectochilus roxburghii* 'Pingbian'

[0075]

[0076] Table 6: Results of the orthogonal experiment on plant hormone induction by adventitious buds of *Anoectochilus pingbianense*

[0077]

[0078]

[0079] As shown in Table 6, the optimal adventitious bud germination rate of *Anoectochilus pingbianensis* was achieved when the plant growth hormone composition was 0.75 mg / L 6-BA, 0.5 mg / L 2,4-D, and 1.25 mg / L NAA.

[0080] 5. Optimization of light conditions for adventitious bud induction culture

[0081] The culture medium consisted of MS medium + 1.5 g / L plant gel + 10 g / L sucrose + 0.4 g / L activated carbon powder + 0.75 mg / L 6-BA, 0.5 mg / L 2,4-D, and 1.25 mg / L NAA. An LED light panel (model 08-50350, manufactured by Dixuan Optoelectronics Technology Co., Ltd.) was used. Five relative intensity ratios of red and blue light were set: red light (1:0), 3:7, 5:5, 7:3, and blue light (0:1). For each ratio, three light intensities were set (40, 80, and 120 μmol / m²). 2 / s). Each experiment was repeated in triplicate. Fluorescent lamps were used as a control, with light intensities of 40, 80, and 120 μmol / m². 2 / s. The photoperiod was 12h / d for all treatments. Six identical mature stem segments were inoculated into each bottle of culture medium of the same quality, with each treatment replicated 10 times. Adventitious bud germination was observed 20 days after inoculation. The results are detailed in Table 7.

[0082] Table 7: Effects of Red and Blue LED Illumination Conditions on the Germination Rate of Adventitious Buds of *Anoectochilus roxburghii* 'Pingbian'

[0083]

[0084] As shown in Table 7, the relative intensity ratio of red light to blue light is 5:5, and the illuminance of both is 120 μmol / m². 2 / s, the adventitious bud germination rate of *Anoectochilus roxburghii* is the best.

[0085] Experimental Example 3: Optimization of Rooting Induction Medium and Culture Conditions

[0086] 1. Plant hormone concentration

[0087] The optimal concentration of plant hormones in the rooting induction medium was determined using an orthogonal design (Table 8). MS basal medium was used, supplemented with 1.5 g / L plant gel and 30 g / L sucrose. Six identical mature stem segments were inoculated into each bottle of medium of equal quality, with each treatment replicated 10 times. Rooting of *Anoectochilus roxburghii* was observed 20 days after inoculation. The results are detailed in Tables 8-9.

[0088] Table 8: Orthogonal experimental design for inducing plant hormones in rooting of *Anoectochilus roxburghii* from Pingbian

[0089]

[0090] Table 9: Results of the orthogonal experiment on inducing plant hormones in rooting of *Anoectochilus roxburghii* var. *pingbianensis*

[0091]

[0092] As shown in Table 9, the addition of 0.3 mg / L 6-BA and 0.1 mg / L 1-BA is optimal for inducing rooting of Anoectochilus pingbianense.

[0093] 2. Basic culture medium

[0094] The optimal basal medium for root induction was determined by using 1 / 2 MS, MS, and 2 MS basal media, supplemented with 1.5 g / L plant gel, 30 g / L sucrose, 0.3 mg / L 6-BA, and 0.1 mg / L IBA. Six identical mature stem segments were inoculated into each bottle of medium of equal quality, with each treatment replicated 10 times. Rooting was observed 20 days after inoculation. The results are detailed in Table 10.

[0095] Table 10: Statistical analysis of rooting rate of *Anoectochilus roxburghii* stem segments under different basal culture media.

[0096]

[0097]

[0098] As shown in Table 10, 1 / 2 MS is the optimal basal culture medium for rooting of Anoectochilus pingbianense.

[0099] 3. Sucrose concentration

[0100] The optimal sucrose concentration for rooting induction medium was determined using 1 / 2 MS basal medium supplemented with sucrose concentrations of 10 g / L, 20 g / L, 30 g / L, and 40 g / L, and plant gel concentrations of 1.5 g / L, 0.3 mg / L 6-BA, and 0.1 mg / L IBA. Six identical mature stem segments were inoculated into each bottle of medium of equal quality, with each treatment replicated 10 times. Rooting was observed 20 days after inoculation. The results are detailed in Table 11.

[0101] Table 11: Statistical analysis of rooting rate of *Anoectochilus roxburghii* stem segments with different sucrose concentrations.

[0102]

[0103] As shown in Table 11, adding 30 g / L sucrose is the optimal method for inducing rooting of Anoectochilus pingbianense.

[0104] 4. Lighting conditions

[0105] The optimal light conditions for rooting induction medium were determined using the following formula: 1 / 2 MS medium + 1.5 g / L plant gel + 30 g / L sucrose + 0.3 mg / L 6-BA and 0.1 mg / L IBA. An LED light panel (model 08-50350, manufactured by Dixuan Optoelectronics Technology Co., Ltd.) was used. Five relative intensity ratios of red and blue light were set: red light (1:0), 3:7, 5:5, 7:3, and blue light (0:1), with three light intensities (40, 80, and 120 μmol / m² / s) for each ratio. Each ratio was replicated three times. A fluorescent lamp was used as a control, with light intensities of 40, 80, ...

[0106] 120 μmol / m² / s. Photoperiod was 12 h / d. Six identical mature stem segments were inoculated into each bottle of culture medium of the same quality, with each treatment replicated 10 times. Rooting of *Anoectochilus roxburghii* was observed 20 days after inoculation. The results are detailed in Table 12.

[0107] Table 12: Effects of Red and Blue LED Illumination Conditions on Rooting Rate of Anoectochilus roxburghii (Golden Thread Orchid)

[0108]

[0109]

[0110] Table 12 shows that the relative intensity ratio of red light to blue light is 3:7, and the illuminance of both is 80 μmol / m². 2 / s, the rooting rate of *Anoectochilus roxburghii* stem segments from Pingbian is the best.

[0111] Experiment Example 4: Optimization of Culture Medium and Culture Conditions for Tissue Culture Seedlings

[0112] 1. Basal culture medium and plant hormones

[0113] The optimal basal culture medium and plant hormones for *Anoectochilus roxburghii* tissue culture seedlings from Pingbian were selected. Referring to commonly used rooting and seedling-strengthening media for *Anoectochilus roxburghii* tissue culture seedlings, 1 / 2 MS and MS basal media were used, supplemented with 1.5 g / L plant gel + 30 g / L sucrose. An orthogonal design scheme for plant hormones was used for the experiment (Table 13). Six stem segments obtained from Experiments 1, 2, and 3 were inoculated into each bottle, with 10 replicates per treatment. The growth of *Anoectochilus roxburghii* tissue culture seedlings from Pingbian were observed four months after inoculation. The results are detailed in Tables 13-14.

[0114] Table 13: Orthogonal experimental design of plant hormones in tissue culture seedlings of *Anoectochilus roxburghii* from Pingbian County

[0115]

[0116] Table 14: Results of orthogonal experiments on plant hormones in tissue culture seedlings of *Anoectochilus roxburghii* from Pingbian under different basal culture media

[0117]

[0118]

[0119] As shown in Table 14, the optimal medium for the growth of tissue culture seedlings of *Anoectochilus roxburghii* from Pingbian is 1 / 2 MS basal medium supplemented with 4.0 mg / L 6-BA and 0.4 mg / L NAA.

[0120] 2. Sucrose concentration

[0121] The optimal sucrose concentration for the tissue culture seedlings of *Anoectochilus roxburghii* from Pingbian was determined using 1 / 2 MS basal medium supplemented with 1.5 g / L plant gel + 4.0 mg / L 6-BA and 0.4 mg / L NAA, and sucrose concentrations of 10 g / L, 30 g / L, and 50 g / L were added respectively. Six stem segments (obtained from Experiments 1, 2, and 3) were inoculated per bottle, with each treatment replicated 10 times. The growth of the *Anoectochilus roxburghii* tissue culture seedlings was observed four months after inoculation. The results are detailed in Table 15.

[0122] Table 15: Statistics on the growth of *Anoectochilus roxburghii* tissue culture seedlings with different sucrose concentrations in Pingbian County

[0123]

[0124] As shown in Table 15, adding 30 g / L of sucrose is most suitable for the growth of tissue culture seedlings of *Anoectochilus roxburghii* from Pingbian.

[0125] 3. Lighting conditions

[0126] Optimized light conditions for tissue culture seedlings of *Anoectochilus roxburghii* from Pingbian were achieved using an optimized culture medium consisting of 1 / 2 MS medium, 1.5 g / L plant gel, 30 g / L sucrose, 4.0 mg / L 6-BA, and 0.4 mg / L NAA. An LED light panel (model 08-50350, manufactured by Dixuan Optoelectronics Technology Co., Ltd.) was used. Five relative intensity ratios of red and blue light were established: red light (1:0), 3:7, 5:5, 7:3, and blue light (0:1). For each ratio, three light intensities (40 μmol / m²) were set. 2 / s, 80μmol / m 2 / s and 120μmol / m 2 / s). Each experiment was repeated in triplicate. A fluorescent lamp was used as a control, with a light intensity of 40 μmol / m². 2 / s, 80μmol / m 2 / s and 120μmol / m 2 / s. The photoperiod was 12h / d. Six stem segments obtained from Experiments 1, 2, and 3 were inoculated into each bottle, with each treatment replicated 10 times. The growth of the *Anoectochilus roxburghii* tissue culture seedlings was observed four months after inoculation. The results are detailed in Table 16.

[0127] Table 16: Effects of Red and Blue LED Illumination Conditions on the Growth of *Anoectochilus roxburghii* Tissue Culture Seedlings from Pingbian

[0128]

[0129] As shown in Table 16, the relative intensity ratio of red light to blue light is 5:5, and the illuminance of both is 120 μmol / m². 2 / s, the tissue culture seedlings of Anoectochilus roxburghii in Pingbian showed the best growth.

[0130] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for tissue culture of *Anoectochilus roxburghii*, characterized in that, The method includes: (1) Stem segment treatment Select wild *Anoectochilus pingbianensis*, remove leaves and roots, rinse, cut stem segments of *Anoectochilus pingbianensis*, soak in alcohol and rinse with sterile water, soak in sodium hypochlorite and rinse with sterile water to obtain processed stem segments. (2) Adventitious bud induction The treated stem segments were inoculated into an induction medium for culture. The induction medium consists of MS basal medium, 0.25 mg / L to 1 mg / L 6-BA, 0.5 mg / L to 2 mg / L of 2,4-D, 0.75 mg / L to 1.5 mg / L of NAA, 10 g / L to 30 g / L of sucrose, It consists of 0.2 g / L to 0.8 g / L of activated carbon powder and 1 g / L to 2 g / L of plant gel; The cultivation was carried out under a light intensity of 40 μmol / m². 2 / s~120μmol / m 2 The experiment was conducted under red and blue light with a relative intensity of (5-7):(3-5) / s; (3) Rooting induction The stem segments obtained in step (2) were inoculated onto a rooting medium for culture. The rooting medium consists of 1 / 2 to 2 MS basal medium, 0.2 mg / L to 0.4 mg / L 6-BA, 0.05 mg / L to 0.15 mg / L IBA, 10 g / L to 40 g / L sucrose and 1.5 g / L plant gel. The cultivation was carried out under a light intensity of 40 μmol / m². 2 / s~120μmol / m 2 The experiment was conducted under red and blue light with a relative intensity of (3-5):(5-7) / s; (4) Tissue culture seedling culture The stem segments obtained in step (3) are inoculated onto tissue culture medium and cultured to obtain tissue culture seedlings with complete roots, stems and leaves; The tissue culture medium consists of 1 / 2 to 1 MS basal medium, 3.0 mg / L to 5.0 mg / L 6-BA, 0.3 mg / L to 0.5 mg / L NAA, 10 g / L to 30 g / L sucrose and 1.5 g / L plant gel. The cultivation was carried out under a light intensity of 40 μmol / m². 2 / s~120μmol / m 2 The experiment was conducted under red and blue light with a relative intensity of (1-7):(0-5) / s.

2. The method according to claim 1, characterized in that, In step (1), the concentration of sodium hypochlorite is 0.1 g / 100 mL, the stem segment is a mature stem segment, and the length of the stem segment is 0.5 cm.

3. The method according to claim 1, characterized in that, In step (2), the induction medium consists of MS basal medium, 0.75 mg / L 6-BA, 0.5 mg / L 2,4-D, 1.25 mg / L NAA, 10 g / L sucrose, 0.4 g / L activated carbon powder and 1.5 g / L plant gel, and the pH of the induction medium is 5.6 to 5.

8.

4. The method according to claim 1, characterized in that, In step (2), the culture temperature is 22℃~26℃, the humidity is 70%, the daily light time is 12h, and the culture time is 15~20 days; The illumination intensity of both red and blue light is 120 μmol / m². 2 / s, relative intensity is 5:

5.

5. The method according to claim 1, characterized in that, In step (3), the rooting medium consists of 1 / 2 MS basal medium, 0.3 mg / L 6-BA, 0.1 mg / L IBA, 30 g / L sucrose and 1.5 g / L plant gel, and the pH of the rooting medium is 5.6 to 5.

8.

6. The method according to claim 1, characterized in that, In step (3), the culture temperature is 22℃~26℃, the humidity is 70%, the daily light exposure time is 12h, and the culture time is 15~20 days.

7. The method according to claim 1, characterized in that, In step (3), the illumination intensity of both the red and blue light is 80 μmol / m². 2 / s, relative intensity is 3:

7.

8. The method according to claim 1, characterized in that, In step (4), the tissue culture medium consists of 1 / 2 MS basal medium, 4.0 mg / L 6-BA, 0.4 mg / L NAA, 30 g / L sucrose and 1.5 g / L plant gel, and the pH of the tissue culture medium is 5.6 to 5.

8.

9. The method according to claim 1, characterized in that, In step (4), the culture temperature is 22℃~26℃, the humidity is 70%, the daily light exposure time is 12h, and the culture time is 4~5 months.

10. The method according to claim 1, characterized in that, In step (4), the illumination intensity of both the red and blue light is 120 μmol / m². 2 / s, relative intensity is 5:5.

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