Method for rapid seedling formation of pholidota tenuifolia seeds

Abstract

The invention discloses a method for rapid seedling formation of pholidota tenuifolia seeds. Mature or nearly mature pholidota tenuifolia pods are used as explants, the vitamin content of MS and 1 / 2 MS basic culture media is improved through a plant tissue technology, and the rapid seed seedling technology is successfully researched and developed. The method has the characteristics of small damage of explants to stock plants, greenness, no toxicity, high disinfection success rate, high propagation coefficient, low cost, short production period and the like, only 120-140 days are needed in the process from seeds to transplanting into seedlings, the production period is shortened by about 60%-75%, and the production efficiency is greatly improved. The method is a key for protecting precious wild plant resources of pholidota tenuifolia, an efficient and innovative technical approach is provided for seedling industrialization of pholidota tenuifolia, the market supply and demand contradiction of pholidota tenuifolia is solved, and yield guarantee is provided for sustainable development and utilization of subsequent three-dimensional greening resources of pholidota tenuifolia. The method is simple to operate and easy to implement, and the used culture medium and reagent are easy to prepare, low in cost and easy to popularize in a large range.

Description

Technical Field

[0001] The present invention belongs to the field of plant biotechnology, and particularly relates to a method for rapid seedling formation of Pholidota cantonensis seeds. Background Art

[0002] Pholidota cantonensis is a perennial herbaceous plant of the genus Pholidota in the Orchidaceae family. It often climbs on rocks, tree trunks, and vines in the wild. Its main production areas are Zhejiang, Jiangxi, Fujian, Taiwan, Hunan, Guangdong, and Guangxi. The whole herb of Pholidota cantonensis is used as medicine, with the effects of nourishing yin and clearing the lung, promoting diuresis and removing stasis, etc., and can treat diseases such as pulmonary tuberculosis, dizziness and headache, cough and hemoptysis, dysentery, etc. Folk commonly use fresh or dried Pholidota cantonensis to make soup as a medicinal diet. Currently, it is found that Pholidota cantonensis mainly contains flavonoids, polysaccharides, terpenoids, phenols, aliphatic compounds, etc. Pholidota cantonensis also has high ornamental value. Its pseudobulbs are like emeralds, with two green leaves, bright and verdant, and a string of white flowers, elegant and unique, making it a new and excellent three-dimensional greening plant.

[0003] Pholidota cantonensis likes humid and shady environments and grows wild on cliffs. Its growth is extremely slow, the seed germination rate is low, and the growth environment is harsh. Under natural conditions, the germination rate of Pholidota cantonensis seeds is low. Usually, only one bud point grows upward, and the growth rate is low. Conventional seedling separation or sowing methods are difficult to breed seedlings. After a large amount of wild Pholidota cantonensis resources have been over-excavated, the population is difficult to recover in the short term, resulting in a sharp reduction in wild Pholidota cantonensis resources, which is close to endangerment. In 2023, Pholidota cantonensis was listed as a key wild plant in Guangdong Province. Therefore, breaking through the breeding technology bottleneck of Pholidota cantonensis and developing a rapid seedling formation technology is not only the key to protecting its precious wild plant resources, but also can provide an efficient and innovative technical approach for its seedling industrialization, solve the market supply and demand contradiction of Pholidota cantonensis, and provide a yield guarantee for its subsequent sustainable development and utilization of resources.

[0004] Plant tissue culture technology is a high-tech biotechnology that has been successfully applied to the industrialization of seedlings of fruit trees, flowers, vegetables, forest trees, traditional Chinese medicines, etc. Obtaining virus-free seedlings through tissue culture rapid propagation is an innovative and rapid technical means. However, due to imperfect disinfection methods, long seed germination time, long culture cycle, high culture cost, etc. in the existing tissue culture and breeding technology of Orchidaceae plants, it takes a long time from seeds to seedlings. Among them, Pholidota cantonensis requires at least more than 400 days; Pholidota chinensis requires 250 - 540 days; Dendrobium officinale requires 200 - 325 days; Cymbidium floribundum requires 240 - 290 days; Arundina graminifolia requires about 255 days; Cymbidium ensifolium requires 330 days; Oncidium requires 270 days; Paphiopedilum requires 232 - 288 days; Cymbidium requires 204 - 240 days; Cattleya requires 275 days; Bulbophyllum requires 515 days; Epidendrum requires 270 days, etc.

[0005] At present, there is a lack of relevant research on the rapid seedling formation technology of Pholidota cantonensis. Through a green, efficient, and non-toxic disinfection method, the present invention improves the seed germination rate, improves the basic medium formula, reduces the culture cost, enhances the strong seedling effect and rooting rate, and establishes a perfect regeneration system for Pholidota cantonensis, shortening the production cycle by about 65%-70%, greatly shortening the production cycle, and improving the production efficiency. The present invention is not only the key to protecting the precious wild plant resources of Pholidota cantonensis, but also can provide an efficient and innovative technical approach for its seedling industrialization, solve the contradiction between market supply and demand, and provide yield guarantee for the sustainable development and utilization of its subsequent three-dimensional greening resources. Summary of the Invention

[0006] The object of the present invention is to provide a method for rapid seedling formation of Pholidota cantonensis seeds, which has little damage to the mother plant, is green, non-toxic, has a high disinfection success rate, a low pollution rate, a high propagation coefficient, a low cost, a fast growth rate, and a short production cycle, solves the problems of long tissue culture cycle and high cost of Pholidota cantonensis in the prior art, and provides an efficient and innovative technical approach for its seedling industrialization.

[0007] The present invention is realized through the following technical solutions:

[0008] A method for rapid seedling formation of Pholidota cantonensis seeds, comprising the following steps:

[0009] (1) Disinfection of explants and seed germination: Select mature or nearly mature fruit pods of Pholidota cantonensis as explants. After disinfection treatment, perform cutting treatment and inoculate them into a seed germination medium for culture. The culture temperature is 25±1°C, the light intensity is 2000-3000 lx, and the light time is 8-12 h / d. The seeds swell and turn green after 12d-15d, and can germinate into the protocorm state in about 40d;

[0010] (2) Induction of adventitious buds from protocorms: Transfer the protocorms obtained in step (1) to an adventitious bud induction medium for culture. The culture temperature is 25±1°C, the light intensity is 2000-3000 lx, and the light is 8-12 h / d. Adventitious cluster buds are induced from the protocorms in about 20d;

[0011] (3) Subculture and proliferation culture of adventitious buds: Cut the adventitious buds induced in step (2) into appropriate-sized cluster buds and then subculture them in an adventitious bud subculture and proliferation medium for adventitious bud proliferation. The culture temperature is 25±1°C, the light intensity is 2000-3000 lx, and the light is 8-12 h / d. The subculture and proliferation cycle is 20d;

[0012] (4) Root induction culture: Cut the adventitious buds that have grown to 2-3 cm and formed pseudobulbs, transfer them to the root induction medium for root culture. The culture temperature is 25±1°C, the light intensity is 2000-3000 lx, the light duration is 8-12 h / d, and roots are induced from the adventitious buds in 10-14 days.

[0013] (5) Transplanting of test-tube plantlets: After root culture, the test-tube plantlets are acclimatized under natural light and temperature conditions, then the medium on the roots of the test-tube plantlets is washed off and transplanted into the cultivation substrate.

[0014] The composition of the seed germination medium is: Modified MS medium + 6-BA 0.5-2.0 mg·L -1 + NAA 0.1-0.4 mg·L -1 + 6% w / v banana puree + sucrose 30 g / L + agar 7.0 g / L, pH 5.8-6.0;

[0015] The composition of the adventitious bud induction medium or adventitious bud subculture and proliferation medium is: Modified MS medium + 6-BA 1.0-4.0 mg·L -1 + NAA 0.2-0.5 mg·L -1 + 3% w / v banana puree + sucrose 30 g / L + agar 7.0 g / L, pH 5.8-6.0;

[0016] The composition of the root induction medium is: Modified 1 / 2 MS medium + IBA 0-1.0 mg·L -1 + NAA 0-1.0 mg·L -1 + activated carbon 0.6 g·L -1 + sucrose 30 g / L + carrageenan 6.0 g / L, pH 5.8-6.0.

[0017] Preferably, the composition of the seed germination medium is: Modified MS medium + 6-BA 2.0 mg·L -1 + NAA 0.1 mg·L -1 + 6% w / v banana puree + sucrose 30 g / L + agar 7.0 g / L, pH 5.8-6.0; The composition of the adventitious bud induction medium is: Modified MS medium + 6-BA 4.0 mg·L -1 + NAA 0.5 mg·L -1 + 3% w / v banana puree + sucrose 30 g / L + agar 7.0 g / L + pH 5.8-6.0; The composition of the adventitious bud subculture and proliferation medium is: Modified MS medium + 6-BA 2.0 mg·L -1 + NAA 0.4 mg·L -1+ 3% w / v banana puree + 30 g / L sucrose + 7.0 g / L agar, pH 5.8 - 6.0; The rooting induction medium consists of: Modified 1 / 2 MS medium + 0.5 mg·L -1 + 0.6 g·L activated carbon -1 + 30 g / L sucrose + 6.0 g / L carrageenan, pH 5.8 - 6.0.

[0018] Preferably, the modified MS medium is: based on the conventional MS basal medium, adjust thiamine hydrochloride (VB1) from 0.1 mg / L to 0.5 mg / L, and nicotinic acid from 0.5 mg / L to 5 mg / L; The modified 1 / 2 MS medium is: based on the conventional 1 / 2 MS basal medium, adjust thiamine hydrochloride (VB1) from 0.1 mg / L to 0.5 mg / L, and nicotinic acid from 0.5 mg / L to 5 mg / L. The formula and preparation method of the conventional MS basal medium and the conventional 1 / 2 MS basal medium can be found in the literature: Huang Xiaomei. Plant Tissue Culture [M]. Chemical Industry Press, 2019.

[0019] Preferably, in step (1), the specific steps of the disinfection treatment are: select mature or nearly mature fruit pods of Pholidota cantonensis as explants, shake the fruit pods of Pholidota cantonensis with 75% (v / v) alcohol for 30 seconds, and then soak them in sterile water. Then put them into a 20% (v / v) sodium hypochlorite solution and soak for 8 minutes. After rinsing 3 times with sterile water, dry the water with sterile filter paper.

[0020] Preferably, in step (1), after the disinfection treatment, in a laminar flow hood, cut the fruit pods with a sterile scalpel, and evenly sprinkle the seeds on the seed germination medium for seed germination culture.

[0021] Preferably, in step (1), the seed germination culture stage requires dark culture for two weeks, and then enters the light culture.

[0022] Preferably, in step (5), the acclimatization is to transfer the obtained regenerated plants together with the culture medium and culture bottle into a greenhouse for acclimatization, and acclimatize without opening the lid for 10 - 14 days.

[0023] Preferably, in step (5), the regenerated plants after acclimatization are transplanted into sphagnum moss substrate, placed in a greenhouse nursery at a temperature of 26°C, a light intensity of 3000 lx, a light duration of 12 h / d, and the humidity is maintained at 70 - 75%. The survival rate can reach 95 - 98% after 15 days.

[0024] Advantages of the present invention:

[0025] The present invention uses mature or nearly mature fruit pods of Pholidota cantonensis as explants, and through plant tissue technology, improves the vitamin content of MS and 1 / 2MS basal media, successfully develops a rapid seedling technology for Pholidota cantonensis, and breaks through the bottleneck of the breeding technology of Pholidota cantonensis. The present invention has the characteristics of small damage to the mother plant by the explant, being green and non-toxic, high disinfection success rate, high propagation coefficient, low cost, short production cycle, etc. It only takes 120 - 140 days from seed to transplanting and seedling formation, and the production cycle is shortened by about 60% - 75%, greatly improving the production efficiency. The present invention is not only the key to protecting the precious wild plant resources of Pholidota cantonensis, but also can provide an efficient and innovative technical approach for its seedling industrialization, solve the market supply and demand contradiction, and provide yield guarantee for the sustainable development and utilization of its subsequent three-dimensional greening resources. The method of the present invention is simple to operate and easy to implement, and the used culture medium and reagents are easy to prepare, with low cost and easy to promote on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the wild plant of Pholidota cantonensis.

[0027] Figure 2 is the process of tissue culture of Pholidota cantonensis of the present invention, wherein, A: seed germination; B: protocorm proliferation; C: seedling rooting; D: transplanting the seedling to sphagnum moss substrate. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0029] Embodiment:

[0030] 1 Materials and Methods

[0031] 1.1 Test Materials

[0032] In this test, mature or nearly mature fruit pods of Pholidota cantonensis were used as test materials, and the fruit pods were collected from Bazi Garden, Danxiashan, Renhua County, Shaoguan City, Guangdong Province.

[0033] 1.2 Test Methods

[0034] 1.2.1 Sterile Treatment of Materials

[0035] After shaking the fruits of Pholidota cantonensis with 75% alcohol by volume for 30 seconds, soak them in sterile water. Then disinfect them in a 20% sodium hypochlorite solution by volume for 8 minutes, rinse them 3 times with sterile water, dry them with sterile filter paper, longitudinally cut the materials with a sterile scalpel, evenly sprinkle the seeds on the medium, and conduct dark culture for two weeks after inoculation. After the seeds germinate, transfer them to interval light culture under a light intensity of 2000 - 3000 lx, and the culture temperature is 25 ± 1°C.

[0036] 1.2.2 Selection of basic medium

[0037] Based on the results of the influence of different basic media on the cycle of different culture stages of Pholidota cantonensis (from seed germination to protocorm, adventitious bud induction, subculture proliferation, and rooting induction stages), select the best basic medium for each stage. The specific screening conditions are as follows:

[0038] From seed germination to protocorm: basic medium + 6 - BA 1.0 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8 - 6.0, temperature 25 ± 1°C, light intensity 2000 - 3000 lx, light duration 8 - 12 h / d;

[0039] Adventitious bud induction: basic medium + 6 - BA 2.0 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8 - 6.0, temperature 25 ± 1°C, light intensity 2000 - 3000 lx, light duration 8 - 12 h / d;

[0040] Subculture proliferation: basic medium + 6 - BA 2.0 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8 - 6.0, temperature 25 ± 1°C, light intensity 2000 - 3000 lx, light duration 8 - 12 h / d;

[0041] Rooting induction: basic medium + IBA 1.0 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH 5.8 - 6.0, temperature 25 ± 1°C, light intensity 2000 - 3000 lx, light duration 8 - 12 h / d.

[0042] According to the characteristics of Orchidaceae plants, the basic media are set as MS medium, modified MS medium, 1 / 2MS medium, modified 1 / 2MS medium, and 1 / 4MS medium. The modified MS medium is based on the conventional MS basic medium, with thiamine hydrochloride (VB1) adjusted from 0.1 mg / L to 0.5 mg / L and nicotinic acid adjusted from 0.5 mg / L to 5 mg / L. The modified 1 / 2MS medium is based on the conventional 1 / 2MS basic medium, with thiamine hydrochloride (VB1) adjusted from 0.1 mg / L to 0.5 mg / L and nicotinic acid adjusted from 0.5 mg / L to 5 mg / L.

[0043] Table 1 Effects of Different Basic Media on the Cycle of Different Culture Stages

[0044]

[0045] The experimental results show that different basic media have a great impact on the cycle of different culture stages of Pholidota cantonensis. As shown in Table 1, the production cycle lengths of Pholidota cantonensis are in the order of: modified MS medium < MS medium < modified 1 / 2MS medium < 1 / 2MS medium < 1 / 4MS medium. Among them, the seeds of Pholidota cantonensis in 1 / 4MS medium stopped growing and failed to enter the subsequent stages, indicating that 1 / 4MS medium is not suitable for its rapid propagation system.

[0046] For the modified MS medium, during the stages of seed germination to protocorm, adventitious bud induction, and subculture proliferation, it takes at least 40 days, 20 days, and 20 days respectively, and the time is significantly shorter than that of other basic media; while for the modified 1 / 2MS medium, the time required for root induction is 10 - 14 days. Therefore, in the selection of basic media, the best choice for the stages of seed germination to protocorm, adventitious bud induction, and subculture proliferation is the modified MS medium, and the best choice for the root induction stage is the modified 1 / 2MS medium. Using the two in combination can greatly shorten the production cycle of the entire Pholidota cantonensis and improve production efficiency.

[0047] 1.2.3 Selection of Plant Growth Regulators and Additives

[0048] On the basis of screening out the best basic medium, different types and concentrations of growth regulators and additives are added according to different culture stages to further screen the most suitable medium formula.

[0049] 1.2.3.1 Seed Germination

[0050] The seeds (the same as those in 1.2.1 above) were evenly sown on the seed germination medium for cultivation. The seed germination medium was as follows: on the modified MS medium, adding 7 g / L of agar, 30 g / L of sucrose, and controlling the pH at 5.8 - 6.0. Different concentrations of 6-benzylaminopurine (6-BA; 0.5, 1.0, 2.0 mg·L -1 ) and naphthaleneacetic acid (NAA; 0.1, 0.2, 0.4 mg·L -1 ) and banana puree (0, 30, 60 g·L -1 ) were added, and the effects of the 3-factor 3-level orthogonal test combinations (A1 - A9) on seed germination were studied. 10 culture dishes were inoculated in each group, with 3 replicates. After 14 days of shading treatment, they were transferred to normal light. The culture conditions were: temperature 25 ± 1 °C, light intensity 2000 - 3000 lx, and light duration 8 - 12 h / d.

[0051] Table 2 Effects of different contents of 6-BA, NAA, and banana puree on seed germination

[0052]

[0053]

[0054] As can be seen from Table 2, the seeds of Pholidota cantonensis can germinate to varying degrees on the media added with different concentrations of 6-BA, NAA, and banana puree. The germination rate of the seeds of Pholidota cantonensis was the highest in A9 ( Figure 2 A), which was 85.37%. The seeds swelled and turned green after 12 days, and could germinate into the protocorm state in 40 days. At this time, the protocorms were green, dark green at the top, and had root hairs. Followed by A8, the germination rate was 80.88%, which was significantly higher than the seed germination rates in other media. The germination rate of A5 was the lowest. The experiment showed that high concentrations of 6-BA and banana puree were beneficial to seed germination. The optimal seed germination medium for Pholidota cantonensis was: modified MS medium + 6-BA 2.0 mg·L-1 + NAA 0.1 mg·L-1 + 6% banana puree + sucrose 30 g / L + agar 7.0 g / L, pH 5.8 - 6.0.

[0055] 1.2.3.2 Induction of adventitious buds and adventitious bud proliferation culture

[0056] The protocorms in the above-mentioned A9 seed germination medium were transferred to the medium for induction of adventitious buds and proliferation culture. The medium was as follows: on the modified MS medium, adding 30 g / L of banana puree, 7 g / L of agar, 30 g / L of sucrose, and adding different concentrations of naphthaleneacetic acid (NAA; 0.2, 0.4, 0.5 mg·L -1 ) and 6-benzylaminopurine (6-BA; 1.0, 2.0, 4.0 mg·L -1 ) and activated carbon (0, 0.5, 1.0 g·L-1 )Plant growth regulators with a 3-factor and 3-level orthogonal combination (B1 - B9). For each treatment, 20 culture bottles were inoculated, with 3 replicates, and 6 clusters of protocorms were inoculated in each bottle. Observe the protocorms, count the proliferation coefficient, and the culture conditions are the same as above. In the medium experiment at the same stage, the adventitious bud induction medium and the adventitious bud subculture proliferation medium were screened out.

[0057] Table 3 Effects of different concentrations of NAA, IBA, and activated carbon on the induction of adventitious buds and the proliferation of adventitious buds

[0058]

[0059] As can be seen from Table 3, the concentrations of 6-BA and NAA can affect the induction of adventitious buds from protocorms and the subculture proliferation coefficient of adventitious buds. The content of activated carbon inhibits the number of adventitious buds to a certain extent, but the seedling plant color of its seedlings is darker than that of other treatment groups. The optimal medium for inducing adventitious buds from the protocorms of Pholidota cantonensis is B3, and the highest proliferation coefficient is 4, and it induces more adventitious buds ( Figure 2 B); The proliferation coefficient of medium B2 can induce green, strong and healthy seedlings, and it is easy to grow pseudobulbs, which is suitable for strengthening seedlings of Pholidota cantonensis before root emergence.

[0060] 1.2.3.3 Rooting culture

[0061] The adventitious buds with similar growth and forming pseudobulbs were divided into individual plants and inoculated on the rooting medium respectively. The rooting medium is a modified 1 / 2MS medium with a 3-factor and 3-level orthogonal combination of different concentrations of naphthaleneacetic acid (NAA; 0, 0.5, 1.0 mg·L -1 ), (IBA; 0, 0.5, 1.0 mg·L -1 ), and activated carbon (0, 0.3, 0.6 g·L -1 ), added with 30 g / L of sucrose and 6 g / L of carrageenan, and carried out rooting culture. For each treatment, 30 culture bottles were inoculated, with 3 replicates, and 10 plants were inoculated in each bottle. Observe the plants, count the number of roots and the rooting rate among different treatment groups, and the culture conditions are the same as above.

[0062] Table 4 Effects of different concentrations of NAA, IBA, and activated carbon on the rooting of proliferated buds

[0063]

[0064] As can be seen from Table 4, a certain concentration of NAA and IBA can promote the rooting of proliferated buds of Pholidota cantonensis, but high concentrations of NAA and IBA both inhibit the rooting of seedlings, while adding an appropriate amount of activated carbon can increase the number of roots and the rooting rate of seedlings of Pholidota cantonensis. The experimental results show that the optimal rooting medium for Pholidota cantonensis is the modified 1 / 2MS medium + IBA 0.5 mg·L -1 + activated carbon 0.6 g·L-1 + 30 g / L of sucrose + 6.0 g / L of carrageenan, pH 5.8 - 6.0; the average number of roots is 3.3, and the rooting rate is 90%. Its roots are thicker than those of other treatment groups, and the number of root hairs is large.

[0065] 1.2.3.4 Acclimatization and transplantation

[0066] Select well - grown and robust tissue - cultured seedlings and place them in the greenhouse for acclimatization (transfer them into the greenhouse together with the culture medium and culture bottle for acclimatization without opening the lid). After 14 days, wash the culture medium on the roots with clean water. After rinsing thoroughly, transplant them into three substrates: sphagnum moss, coconut coir, and bark, and culture them at room temperature. There are 10 seedling boxes for each treatment, repeated 3 times. 10 plants are inoculated in each seedling box, and water is poured once every 3 days. The survival rate is counted 15 days after transplantation. The culture conditions are: temperature 26 °C, light intensity 3000 lx, light duration 12 h / d, and humidity is maintained at 70 - 75%.

[0067] After 15 days of transplanting Pholidota cantonensis, the survival rates of sphagnum moss and coconut coir are significantly higher than that of bark. The transplant survival rates with sphagnum moss and coconut coir as the transplant substrates are 98.3% and 95% respectively; while the survival rate of bark is the lowest, only 56.6%. The experimental results show that when transplanting Pholidota cantonensis into sphagnum moss with higher moisture - retaining performance, the survival rate of seedlings is higher.

Claims

1. A method for rapid seedling formation of Pholidota cantonensis Rolfe var. bella (Rolfe) Tang et Wang, characterized in that, It includes the following steps: (1) Explant disinfection and seed germination: Select the fruit pods of Pholidota cantonensis Rolfe as explants. After disinfection treatment, they are cut and inoculated into the seed germination medium for seed germination culture to form protocorms. The composition of the seed germination medium is: Modified MS medium + 6-BA 0.5 - 2.0 mg·L -1 + NAA 0.1 - 0.4 mg·L -1 + 6% w / v banana puree + sucrose 30 g / L + agar 7.0 g / L, pH 5.8 - 6.0; (2) Induction of adventitious buds from protocorms: Transfer the protocorms obtained in step (1) to an adventitious bud induction medium for induction culture to form adventitious bud clusters; the composition of the adventitious bud induction medium is: modified MS medium + 6-BA 1.0 - 4.0 mg·L -1 + NAA 0.2 - 0.5 mg·L -1 + 3% w / v banana puree + sucrose 30 g / L + agar 7.0 g / L, pH 5.8 - 6.0; (3) Subculture and proliferation culture of adventitious buds: Cut the adventitious cluster buds obtained in step (2) and place them in an adventitious bud subculture and proliferation medium for proliferation culture; the composition of the adventitious bud subculture and proliferation medium is: modified MS medium + 6-BA 1.0 - 4.0 mg·L -1 + NAA 0.2 - 0.5 mg·L -1 + 3% w / v banana puree + sucrose 30 g / L + agar 7.0 g / L, pH 5.8 - 6.0; (4) Root induction culture: Cut the adventitious buds that form pseudobulbs and transfer them into the root induction medium for root culture to obtain test-tube seedlings; the composition of the root induction medium is: modified 1 / 2MS medium + IBA 0 - 1.0 mg·L -1 + NAA 0 - 1.0 mg·L -1 + activated carbon 0.6 g·L -1 + sucrose 30 g / L + carrageenan 6.0 g / L, pH 5.8 - 6.0; (5) Transplanting test-tube plantlets: The test-tube plantlets after rooting culture are acclimatized under natural light and temperature, then the root medium is washed off, and they are transplanted into the substrate. The improved MS medium is: on the basis of the conventional MS basal medium, thiamine hydrochloride is adjusted from 0.1 mg / L to 0.5 mg / L, and nicotinic acid is adjusted from 0.5 mg / L to 5 mg / L; the improved 1 / 2MS medium is: on the basis of the conventional 1 / 2MS basal medium, thiamine hydrochloride is adjusted from 0.1 mg / L to 0.5 mg / L, and nicotinic acid is adjusted from 0.5 mg / L to 5 mg / L.

2. The method according to claim 1, wherein The composition of the seed germination medium is: improved MS medium + 6-BA 2.0 mg·L -1 + NAA 0.1 mg·L -1 + 6% w / v banana puree + sucrose 30 g / L + agar 7.0 g / L, pH 5.8 - 6.0; The composition of the adventitious bud induction medium is: improved MS medium + 6-BA 4.0 mg·L -1 + NAA 0.5 mg·L -1 + 3% w / v banana puree + sucrose 30 g / L + agar 7.0 g / L, pH 5.8 - 6.0; The composition of the adventitious bud subculture and proliferation medium is: improved MS medium + 6-BA 2.0 mg·L -1 + NAA 0.4 mg·L -1 + 3% w / v banana puree + sucrose 30 g / L + agar 7.0 g / L, pH 5.8 - 6.0; The composition of the rooting induction medium is: improved 1 / 2MS medium + IBA 0.5 mg·L -1 + activated carbon 0.6 g·L -1 + sucrose 30 g / L + carrageenan 6.0 g / L, pH 5.8 - 6.

0.

3. The method according to claim 1 or 2, characterized in that, In step (1), the specific steps of the disinfection treatment are: Select mature or nearly mature fruit pods of Pholidota cantonensis as explants. After shaking the fruit pods with 75% (v / v) alcohol for 30 seconds, soak them in sterile water, then put them into a 20% (v / v) sodium hypochlorite solution and soak for 8 minutes. After rinsing 3 times with sterile water, dry the moisture with sterile filter paper.

4. The method according to claim 1 or 2, characterized in that In step (1), after the disinfection treatment, in the ultra-clean workbench, cut the fruit pods with a sterile scalpel, and evenly sprinkle the seeds on the seed germination medium for seed germination culture.

5. The method according to claim 1 or 2, characterized in that, In step (1), the seed germination culture requires dark culture for two weeks, and then enters light culture.

6. The method according to claim 1 or 2, characterized in that, In step (4), the adventitious buds forming pseudobulbs grow to 2 - 3 cm.

7. The method according to claim 1 or 2, characterized in that, The conditions for seed germination culture in step (1) are: temperature 25 ± 1 °C, light 2000 - 3000 lx, light time 8 - 12 h / d; the conditions for induction culture in step (2) are: temperature 25 ± 1 °C, light 2000 - 3000 lx, light 8 - 12 h / d; the conditions for proliferation culture in step (3) are: temperature 25 ± 1 °C, light 2000 - 3000 lx, light 8 - 12 h / d; the conditions for rooting culture in step (4) are: temperature 25 ± 1 °C, light 2000 - 3000 lx, light 8 - 12 h / d.

8. The method according to claim 1 or 2, characterized in that, In step (5), the acclimatization is to transfer the obtained regenerated plants together with the medium and culture bottles into the greenhouse, and acclimatize without opening the lid for 10 - 14 d.

9. The method according to claim 1 or 2, characterized in that, In step (5), the transplantation into the substrate is specifically: transplant into sphagnum moss substrate, place it in the greenhouse nursery, temperature 26 °C, light 3000 lx, light time 12 h / d, and keep the humidity at 70 - 75%.

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