A culture promoter for in vitro culture of caulis of kepitai iron and its application

Abstract

The application discloses a kind of in vitro culture accelerant of kepler ironland stem tip and application thereof.The accelerant contains trans-zeatin riboside, brassinolide and naphthalene acetic acid with mass ratio of 2.0-3.0:1.0-1.5:0.1-0.5, which can effectively improve the in vitro culture effect of kepler ironland stem tip.The application applies the accelerant to the in vitro rapid propagation of kepler ironland stem tip, and the specific method is as follows: cutting the stem tip of kepler ironland plant for disinfection culture; after exposing leaf primordium and apical growth point of explant, inoculating in adventitious bud culture medium to induce culture to obtain adventitious bud; inoculating the adventitious bud in proliferation culture medium to culture to produce multiple shoots; taking out the multiple shoots and placing in strong seedling culture medium for strong seedling culture to complete propagation; the adventitious bud culture medium, proliferation culture medium and strong seedling culture medium contain the accelerant.The in vitro rapid propagation technology of kepler ironland stem tip based on the accelerant effectively improves the propagation success rate and efficiency of kepler ironland.

Description

TECHNICAL FIELD

[0001] The present application relates to a plant tissue culture promoter and its application, in particular to a Tillandsia capitata stem tip in vitro culture promoter and its application. BACKGROUND

[0002] Tillandsia capitata is a herbaceous plant of Tillandsia in Bromeliaceae, and its propagation mainly relies on seed propagation and stolon propagation, but the propagation speed of the two ways is relatively slow, which limits its popularization and application. Tissue culture and rapid propagation technology can accelerate its propagation speed, and the current reports on tissue culture and rapid propagation of Tillandsia plants mainly involve seeds as explants, and a small amount of literature about daughter plants as explants, but there is no report on stem tip in vitro culture.

[0003] The plant growth regulators for promoting Tillandsia tissue culture in the prior art include gibberellin, indole-3-acetic acid, benzylaminoadenine, etc., and a culture method for inducing air pineapple by tissue culture is disclosed in Chinese patent CN 104094844 A, which improves the tissue culture effect of Tillandsia capitata daughter plants by adding gibberellin, indole-3-acetic acid, benzylaminoadenine and other plant growth regulators in the MS culture medium, but the method does not disclose how to culture the stem tip tissue of other varieties of Tillandsia, and it is not known how to select and match plant growth regulators to improve the tissue culture and rapid propagation effect of Tillandsia capitata. SUMMARY

[0004] The purpose of the present application is to provide a Tillandsia capitata stem tip in vitro culture promoter to solve the problem of how to promote the tissue culture and rapid propagation effect of Tillandsia capitata. Another purpose of the present application is to provide a method for improving the Tillandsia capitata stem tip in vitro rapid propagation by using the promoter to solve the problem of how to in vitro rapidly propagate Tillandsia capitata.

[0005] The Tillandsia capitata stem tip in vitro culture promoter comprises trans-zeatin riboside, brassinolide and naphthalene acetic acid in a mass ratio of 2.0-3.0:1.0-1.5:0.1-0.5.

[0006] The present application screens and combines trans-zeatin riboside, brassinolide and naphthalene acetic acid, three kinds of plant growth regulators, for the stem tip in vitro culture method of Tillandsia capitata, which can improve the tissue culture and rapid propagation effect and speed of Tillandsia capitata at an appropriate working concentration.

[0007] The above-mentioned promoter is applied in the Tillandsia capitata stem tip in vitro rapid propagation, and the specific method comprises the following steps:

[0008] (1) cut the stem tip of the Kipte iron plant as an explant, and disinfect and culture the explant;

[0009] (2) after exposing the leaf primordium and the apical growth point of the explant, inoculate in the adventitious bud culture medium to induce and culture to obtain the adventitious bud;

[0010] (3) inoculate the adventitious bud in the proliferation culture medium to culture to generate the clustered bud;

[0011] (4) take out the clustered bud and put it into the strong seedling culture medium to culture to obtain the tissue culture seedling, and cultivate the tissue culture seedling to complete the propagation; at least one of the adventitious bud culture medium, the proliferation culture medium and the strong seedling culture medium contains the promoter in claim 1.

[0012] The application successfully constructs the in-vitro rapid propagation technology system of the Kipte iron plant stem tip by adding the above-mentioned promoter in the culture medium in the tissue culture process.

[0013] Preferably, in step (1), the disinfection and culture method is: 75% alcohol treatment for 40s, and then 0.2% HgCl2 treatment for 10-15min, or 75% alcohol treatment for 60s, and then 0.2% HgCl2 treatment for 10-15min.

[0014] Preferably, in step (2), the adventitious bud culture medium contains 2.5mg / L trans-zeatin riboside, 1.0-1.5mg / L brassinolide and 0.1mg / L naphthalene acetic acid, or 3.0mg / L trans-zeatin riboside, 0.5-1.5mg / L brassinolide and 0.1mg / L naphthalene acetic acid.

[0015] Preferably, in step (3), the proliferation culture medium contains 2.0mg / L trans-zeatin riboside, 1.0-1.5mg / L brassinolide and 0.1mg / L naphthalene acetic acid, or 2.5mg / L trans-zeatin riboside, 1.0-1.5mg / L brassinolide and 0.1mg / L naphthalene acetic acid, or 2.0-2.5mg / L trans-zeatin riboside, 1.5mg / L brassinolide and 0.2mg / L naphthalene acetic acid.

[0016] Preferably, in step (4), the strong seedling culture medium contains 1.0mg / L trans-zeatin riboside, 0.5-1.0mg / L brassinolide and 0.5mg / L naphthalene acetic acid, or 1.0mg / L trans-zeatin riboside, 1.0mg / L brassinolide and 0.2mg / L naphthalene acetic acid.

[0017] Preferably, the adventitious bud culture medium, the proliferation culture medium and the strong seedling culture medium are all MS culture mediums.

[0018] Preferably, in step (2), the induction culture method is as follows: after inoculation, the culture is carried out in a sterile room at a temperature of 20±2℃ for 4-5 days in the dark, and then cultured at a temperature of 25±2℃, a light intensity of 1500-2000 lx, and a light duration of 10h / d.

[0019] Preferably, in step (3), the method for cultivating the bud clusters is as follows: after inoculation, the seedlings are cultivated at a temperature of 25±2℃, a light intensity of 1500-2000 lx, and a light duration of 12h / d; in step (4), the method for cultivating strong seedlings is as follows: the seedlings are cultivated at a temperature of 25±2℃, a light intensity of 2000-2500 lx, and a light duration of 12h / d; the method for cultivating tissue culture seedlings is as follows: when the tissue culture seedlings reach a height of 3-4cm, they are removed from the bottle for cultivation. Before cultivation, the triangular bottles containing the tissue culture seedlings are placed in a greenhouse for 2 days, and then the bottle caps are loosened and left for 7 days. The culture medium is removed, and the tissue culture seedlings are planted on the corresponding cultivation substrate and placed in a shaded greenhouse with a shading rate of 50%-70%. The seedlings are sprayed once in the morning and once in the evening, the temperature is controlled at 25-30℃, and nutrient solution is sprayed once every 7 days. Ventilation is maintained.

[0020] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention can effectively improve the adventitious bud induction, proliferation culture, and seedling vigor effects during the rapid propagation of Cape Tillandsia via shoot tip in vitro. The improvement effect of tissue culture is significantly better than that of existing plant growth regulator compositions. Based on this invention, the rapid propagation technology of Cape Tillandsia via shoot tip in vitro can effectively improve the propagation success rate and efficiency of Cape Tillandsia, reduce propagation costs, and is suitable for large-scale and efficient propagation of Cape Tillandsia. Detailed Implementation

[0021] The technical solution of the present invention will be further described below.

[0022] Example 1: A shoot tip in vitro culture promoter for *Tillandsia pulvinata*, composed of trans-zeatin nucleoside, brassinolide, and naphthaleneacetic acid in a mass ratio of 2.0-3.0:1.0-1.5:0.1-0.5. The specific ratio and concentration can be adapted to actual application requirements.

[0023] The following method is used for rapid in vitro propagation of Cape Tillandsia using the above-mentioned promoters:

[0024] 1. Test materials

[0025] The test materials were provided by the production base of Jiangsu Slow Life Ecological Agriculture Co., Ltd. Uniformly growing Cape Tillandsia plants were selected for the experiment, and the top 0.2-0.3 cm of the stem tip was cut with scissors as explants.

[0026] 2. Methods

[0027] 2.1 Disinfection treatment

[0028] The explants of K. ferrum were put into the triangular flask, covered with gauze, and washed repeatedly under running water for 3-4 h. Then, an appropriate amount of detergent and 2 / 3 tap water were added, and the explants were shaken repeatedly for 0.5 h. Then, the explants were washed with pure water until no foam was produced. After washing, the explants were moved to a clean bench for the next sterilization treatment.

[0029] The sterilization treatment is shown in Table 1. For example, 75% alcohol 20 s + 0.2% HgCl2 25 min means that the explants were first sterilized with 75% alcohol for 20 seconds, and then sterilized with 0.2% HgCl2 for 5 minutes. Other sterilization treatments were performed in the same way. After each sterilization treatment, the explants were washed repeatedly with sterile water for at least 3 times. Finally, the explants were washed with sterile water until completely sterilized, and then the water on the surface of the explants was absorbed with sterilized paper. The explants were inoculated on 1 / 2MS medium. After 10 days, the appearance of the explants was observed, and the contamination rate was recorded.

[0030] Results of different sterilization treatments:

[0031] After different sterilization treatments, the appearance and contamination of K. ferrum stem tips were different, as shown in Table 1. In treatments 1, 2, 3, and 10, the explants were heavily contaminated, with contamination rates of 100.0%, 96.7%, 90.0%, and 83.3%, respectively. In treatments 4, 7, 11, 12, and 13, the explants were moderately contaminated, with contamination rates of 70.0%, 50.0%, 63.3%, 60.0%, and 46.7%, respectively. In treatments 5, 6, 8, and 9, the explants were lightly contaminated, with contamination rates of 30.0%, 16.7%, 13.3%, and 10.0%, respectively. Comparing treatments 5, 6, 8, and 9, although treatment 9 had the lowest contamination rate, the explants were not green or yellow-green, but black, which affected the subsequent bud induction. In treatments 5, 6, and 8, the explants were green or yellow-green, without browning and blackening, which provided a good foundation for the subsequent bud induction. In summary, for the sterilization of K. ferrum stem tips as explants, appropriate sterilization liquid and sterilization time should be selected. In this study, the appropriate sterilization treatment was 75% alcohol treatment for 40 s + 0.2% HgCl2 treatment for 10-15 min or 75% alcohol treatment for 60 s + 0.2% HgCl2 treatment for 10-15 min, which resulted in low contamination rate and green or yellow-green explants.

[0032] Table 1 Results of different sterilization treatments

[0033]

[0034] 2.2 Adventitious bud induction

[0035] After 10 days of culture, the apices of K. ferrum were peeled off layer by layer with forceps, and the peripheral hair and young leaves were peeled off until the leaf primordia and the apical growing points were exposed. Then the apices were inoculated into the corresponding adventitious bud culture medium. The medium formula is shown in Table 2. Each medium formula is based on MS medium with different types and concentrations of plant growth regulators. Among them, tZR is trans-zeatin riboside, BR is brassinolide, NAA is naphthalene acetic acid, BA is benzylaminopurine, GAs is gibberellin, and IAA is indole-3-acetic acid. After inoculation, the culture was carried out in a sterile room at a temperature of 20±2℃ in the dark for 4-5 days, and then at a temperature of 25±2℃ under illumination of 1500-2000lx for 10h / d. The survival rate was counted after 20 days, and the induction of adventitious buds was observed and recorded after 50 days. The induction rate of adventitious buds and the proliferation coefficient of adventitious buds were counted. The survival rate (%) = (the number of surviving apices / the number of inoculated apices) x 100, the induction rate of adventitious buds (%) = (the number of apices inducing adventitious buds / the number of inoculated apices) x 100, and the proliferation coefficient of adventitious buds = the number of induced adventitious buds / the number of inoculated apices.

[0036] Table 2 Induction of adventitious buds in different media

[0037]

[0038]

[0039] Note: tZR2.0+BR0.5+NAA0.1 means that the final concentration of tZR is 2.0 mg / L, the final concentration of BR is 0.5 mg / L, and the final concentration of NAA is 0.2 mg / L. Other formulas are similar.

[0040] Induction of adventitious buds from apices:

[0041] After 20 days of culture, the apical growing points of the apices gradually turned green and sprouted, and the survival rates were not significantly different under different media. The induction of adventitious buds from the apices under different treatments after 50 days is shown in Table 2. Among them, the induction rates of adventitious buds of treatments 5, 6, 7, 8 and 9 were high, 88-92%, and the proliferation coefficients of adventitious buds were also high, 3.75, 3.82, 3.70, 3.90 and 3.87 respectively, and there was no significant difference in the induction rate and proliferation coefficient of adventitious buds among the five treatments. The induction rates and proliferation coefficients of adventitious buds of other treatments were lower. Therefore, the suitable medium for inducing adventitious buds from the apices of K. ferrum is MS+tZR 2.5 mg / L+BR (1.0-1.5 mg / L)+NAA 0.1 mg / L or MS+tZR 3.0 mg / L+BR (0.5-1.5 mg / L)+NAA 0.1 mg / L. The survival rates, induction rates of adventitious buds and proliferation coefficients of adventitious buds of treatments 14-23 were significantly lower than those of treatments 5-9.

[0042] 2.3 Proliferation culture

[0043] After the suitable adventitious bud medium was selected to induce adventitious buds, the adventitious buds were taken out and put into the proliferation medium to continue to grow, the purpose was to promote the proliferation of buds and produce more multiple buds. The culture conditions were: temperature 25±2℃, illumination 1500-2000lx, illumination time 12h / d. The proliferation medium formula treatment was shown in Table 3, each medium formula was added with different types and different concentrations of plant growth regulators on the basis of MS medium, tZT was trans-zeatin, BR was brassinolide, and NAA was naphthalene acetic acid. After 50d, the growth of multiple buds was observed, and the multiple bud proliferation coefficient was counted. Proliferation coefficient = number of multiple buds / number of adventitious buds inoculated.

[0044] Table 3 The induction of multiple buds by different proliferation culture

[0045]

[0046]

[0047] Note: The growth of seedling buds was indicated by robustness and color; robustness: ++++ robust, +++ relatively robust, ++ normal growth, + thin and tender; color: *** dark green, ** green, * yellow green. Glassification: △△△ serious, △△ general, △ no glassification.

[0048] The results of proliferation culture:

[0049] The growth conditions of the shoots were different under different proliferation media, as shown in Table 3. The proliferation coefficients of the shoots induced by treatments 2, 3, 5, 6, 11 and 13 were higher, being 3.50, 3.57, 3.75, 3.82, 3.44 and 3.30 respectively, and the growth conditions of the shoots induced were also good, being healthy, dark green or green in color, and without vitrification. The proliferation coefficients of treatments 7, 8, 9, 10 and 12 were not low, being between 2.02 and 2.80, but the growth of the shoots induced was poor, with serious vitrification. In summary, the suitable proliferation medium for inducing the shoots of K. ferrum was MS + tZT 2.0 mg / L + BR (1.0-1.5) + NAA 0.1 mg / L or MS + tZT 2.5 mg / L + BR (1.0-1.5) mg / L + NAA 0.1 mg / L or MS + tZT (2.0-2.5 mg / L) + BR 1.5 mg / L + NAA 0.2 mg / L. The shoots were inoculated on the selected good proliferation medium, subcultured every 50 d, and after 3 times of continuous subculture, the seedlings were cultured. The proliferation coefficients and the growth conditions of the shoots of treatments 14-23 were also not as good as those of treatments 2, 3, 5, 6, 11 and 13.

[0050] 2.4 Seedling culture

[0051] The shoots were taken out and placed in different seedling culture media, and the culture conditions were as follows: temperature 25 ± 2°C, light 2000-2500 lx, and light time 12 h / d. The seedling culture medium formula treatments are shown in Table 4. Each medium formula was MS medium supplemented with different types and concentrations of plant growth regulators, tZT was trans-zeatin, BR was brassinolide, and NAA was naphthalene acetic acid. The growth of the seedlings was observed after 60 d.

[0052] The growth conditions of the seedlings were different under different seedling culture media, as shown in Table 4. The heights of the seedlings of treatments 3, 3, 5 and 6 were higher, being 3.2 cm, 3.7 cm, 3.6 cm and 4.0 cm respectively, and the growth conditions of the seedlings were good, being healthy and high in degree of robustness, and green or dark green in color. The heights of the seedlings of treatments 11, 12 and 13 were not low, being 2.3-2.8 cm, but the growth conditions of the seedlings were not good, being thin and yellowish green. In summary, in order to promote the vigorous growth of the seedlings of K. ferrum, suitable seedling culture media should be used. It was found in this study that the suitable seedling culture medium formula for K. ferrum tissue culture was MS + tZT 1.0 mg / L + BR (0.5-1.0) mg / L + NAA 0.5 mg / L or MS + tZT 1.0 mg / L + BR (0.5-1.0) mg / L + NAA 0.5 mg / L or MS + tZT 1.0 mg / L + BR (0.5-1.0) mg / L + NAA 0.5 mg / L.

[0053] MS + tZT 1.0 mg / L + BR 1.0 mg / L + NAA 0.2 mg / L. The plant height and seedling growth status of the comparison treatments 13-22 were obviously inferior to those of treatments 3, 4, 5 and 6.

[0054] Table 4 Test results of seedling culture

[0055]

[0056] Note: The seedling growth status is indicated by the robustness and color; robustness: ++++ robust, +++ relatively robust, ++ normal growth, + weak and tender; color: *** dark green, ** green, * yellow-green.

[0057] 3. Conclusion

[0058] 1) Stem tip disinfection treatment: In order to disinfect the Caput stem tips as explants, appropriate disinfectant and disinfection time should be selected. In this study, the appropriate disinfection treatment was 75% alcohol for 40 s + 0.2% HgCl2(10-15 min) or 75% alcohol for 60 s + 0.2% HgCl2(10-15 min), with low explant contamination rate and green or yellow-green color.

[0059] 2) Induction of adventitious buds: The suitable medium for inducing adventitious buds of Caput stem tips was MS + tZR 2.5 mg / L + BR (1.0-1.5 mg / L) + NAA 0.1 mg / L or MS + tZR 3.0 mg / L + BR (0.5-1.5 mg / L) + NAA 0.1 mg / L.

[0060] 3) Induction of proliferation culture of multiple shoots: The suitable medium for inducing proliferation culture of multiple shoots of Caput stem tips was MS + tZT 2.0 mg / L + BR (1.0-1.5) mg / L + NAA 0.1 mg / L or MS + tZT 2.5 mg / L + BR (1.0-1.5) mg / L + NAA 0.1 mg / L or MS + tZT (2.0-2.5 mg / L) + BR 1.5 mg / L + NAA 0.2 mg / L. The multiple shoots were inoculated on the selected excellent proliferation medium formula every 50 d for subculture, and after continuous subculture for 3 times, seedling culture was performed.

[0061] 4) Seedling culture: The suitable medium formula for Caput stem tip culture was MS + tZT 1.0 mg / L + BR (0.5-1.0) mg / L + NAA 0.5 mg / L or MS + tZT 1.0 mg / L + BR 1.0 mg / L + NAA 0.2 mg / L, with healthy growth of seedlings, green or dark green color and height of 3.2-4.0 cm.

[0062] 5) Cultivation of the tissue culture seedlings: when the height of the tissue culture seedlings reaches 3-4 cm, they can be taken out of the bottle for cultivation. Before cultivation, the tissue culture seedlings in the triangular bottle are placed in a greenhouse for 2 days, then the bottle cap is loosened and placed for 7 days, after which the culture medium is washed and the tissue culture seedlings are planted on the corresponding cultivation substrate. In a 32-hole hole tray, a substrate of orchid stone: pine bark: vermiculite = 3:1:1 is laid (wherein the size of the orchid stone is 3-6 mm, the size of the fermented and decomposed pine bark is 6-9 mm, and the orchid stone, pine bark and vermiculite are fully mixed according to a certain volume ratio), and the substrate laid in each hole reaches 3 / 4 of the hole depth. The tissue culture seedlings can be placed on the cultivation substrate.

[0063] Cultivation management measures: light: in a shading greenhouse with a shading rate of 50%-70%. Water: automatic spraying once in the morning at 7-8 o'clock and in the evening at 6-7 o'clock, until the plant is completely wet. Temperature: the automatic temperature control device controls the temperature at 25-30℃. Fertilization: spraying once every 7 days, spraying nutrient solution in the evening at 6-7 o'clock, and the nutrient solution is based on Hoagland nutrient solution with GA3, N, P, K concentrations of 50, 180, 100, 120 mg / l respectively. Ventilation: good ventilation, open the ventilation curtain and water curtain for 2 hours at noon every day.

[0064] The survival rate of the tissue culture seedlings reaches more than 98%, and the growth is healthy and vigorous.

[0065] Example 2: Using the preferred formula and tissue culture method in Example 1 to carry out stem tip tissue culture of different varieties of iron orchids:

[0066] The stem tip disinfection treatment is 75% alcohol for 40s + 0.2% HgCl2 (10-15min).

[0067] The medium for inducing adventitious buds from stem tips is: MS medium + tZR 3.0 mg / L + BR 1.0 mg / L + NAA 0.1 mg / L.

[0068] The proliferation medium for inducing multiple shoots is: MS medium + tZT 2.5 mg / L + BR 1.5 mg / L + NAA 0.1 mg / L.

[0069] The medium for tissue culture of strong seedlings is: MS medium + tZT 1.0 mg / L + BR 1.0 mg / L + NAA 0.5 mg / L.

[0070] The iron orchid varieties are Baowang iron orchid, small elf iron orchid, bulb iron orchid, Vittina iron orchid, and Beckley iron orchid, and the tissue culture results are as follows:

[0071] Table 5 Effect of preferred formula on tissue culture of different varieties of iron orchids

[0072]

[0073] As shown by the results in Table 5, the effect of the accelerators in the application on different varieties of iron orchid tissue culture is quite different, and the effect of the accelerators in the application on the tissue culture propagation of Cape iron orchid is obviously better than that on the tissue culture propagation of the other varieties, and is more suitable for the tissue culture rapid propagation of Cape iron orchid.

Claims

1. A promoter for in vitro culture of *Tillandsia rapa* shoot tips, characterized in that, The accelerator is composed of 2.0-3.0 mg / L trans-zeatin riboside, 1.0-1.5 mg / L brassinolide, 0.1-0.2 mg / L naphthalene acetic acid, and is used for adventitious bud induction medium of stem tip explants of Cephalotaxus fortunei plant; the adventitious bud induction medium is MS medium as basic medium, and the accelerator is added as active ingredient.

2. The use of the accelerant according to claim 1 in the in vitro rapid propagation of shoot tips of Kalpatta ironwood, characterized in that, The method comprises the following steps: (1) cutting stem tips of Cephalotaxus fortunei plant as explants, and disinfecting and culturing the explants; (2) inoculating the explants in the adventitious bud induction medium after exposing leaf primordium and apical growth point of the explants to induce and culture to obtain adventitious buds; (3) inoculating the adventitious buds in the proliferation medium to culture to generate clumps; (4) taking the clumps and placing them in the strong seedling culture medium to culture to obtain tissue culture seedlings, and cultivating the tissue culture seedlings to complete the propagation; The adventitious bud induction medium is MS medium as basic medium, and the accelerator composed of 2.5-3.0 mg / L trans-zeatin riboside, 1.0-1.5 mg / L brassinolide and 0.1-0.2 mg / L naphthalene acetic acid is added as active ingredient; the proliferation medium is MS medium as basic medium, and 2.0 mg / L trans-zeatin, 1.0-1.5 mg / L brassinolide and 0.1 mg / L naphthalene acetic acid, or 2.5 mg / L trans-zeatin, 1.0-1.5 mg / L brassinolide and 0.1 mg / L naphthalene acetic acid, or 2.0-2.5 mg / L trans-zeatin, 1.5 mg / L brassinolide and 0.2 mg / L naphthalene acetic acid are added as active ingredient; the strong seedling culture medium is MS medium as basic medium, and 1.0 mg / L trans-zeatin, 0.5-1.0 mg / L brassinolide and 0.5 mg / L naphthalene acetic acid, or 1.0 mg / L trans-zeatin, 1.0 mg / L brassinolide and 0.2 mg / L naphthalene acetic acid are added as active ingredient.

3. Use according to claim 2, characterized in that, In step (1), the disinfecting and culturing method is: 75% alcohol treatment for 40 s, and then 0.2% HgCl2 treatment for 10-15 min, or 75% alcohol treatment for 60 s, and then 0.2% HgCl2 treatment for 10-15 min.

4. Use according to claim 2, characterized in that, In step (2), the active ingredients in the adventitious bud induction medium are 2.5 mg / L trans-zeatin riboside, 1.0-1.5 mg / L brassinolide and 0.1 mg / L naphthalene acetic acid, or 3.0 mg / L trans-zeatin riboside, 1.0-1.5 mg / L brassinolide and 0.1 mg / L naphthalene acetic acid.

5. Use according to claim 2, characterized in that, In step (2), the induction culture method is: after inoculation, in the sterile room, the temperature is 20±2 ℃, and dark culture is carried out for 4-5 d, and then culture is carried out at the temperature of 25±2 ℃, the illumination of 1500-2000 lx and the illumination time of 10 h / d.

6. Use according to claim 2, characterized in that, In step (3), the method for culturing the cluster buds is: culturing at a temperature of 25±2 ℃, illumination of 1500-2000lx, and illumination time of 12h / d after inoculation; in step (4), the method for culturing the strong seedlings is: culturing at a temperature of 25±2 ℃, illumination of 2000-2500lx, and illumination time of 12h / d; the method for cultivating the tissue culture seedlings is: taking the tissue culture seedlings out of the bottle for cultivation when the height of the tissue culture seedlings reaches 3-4cm, placing the triangular bottle containing the tissue culture seedlings in a greenhouse for 2d before the cultivation, then loosening the bottle cap and placing for 7d, removing the culture medium, planting the tissue culture seedlings on the corresponding cultivation substrate, placing in a shading greenhouse with a shading rate of 50%-70%, spraying once in the morning and once in the evening, controlling the temperature at 25-30℃, and spraying the nutrient solution once every 7d, and keeping ventilation.

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