CN117598204B - Yunnan Hanxiao tissue culture and rapid propagation method and application - Google Patents

Abstract

The present invention relates to a plant groupThe technical field of culture, discloses a tissue culture and rapid propagation method and application of michelia yunnanensis, and the method comprises the following steps: the explant is disinfected, the disinfected explant is inoculated into a cluster bud induction culture medium for culture, the cluster buds induced by the primary culture are inoculated into a secondary proliferation culture medium for culture, and the cluster buds with the secondary proliferation value are inoculated into a rooting culture medium for culture. NAA, 2-iP and ZT with certain concentration are added in an induction culture medium, and the induction rate is improved to more than 60 percent; the secondary proliferation culture medium changes NAA into IBA, and combines ZT and BR with certain concentration to stabilize proliferation coefficient above 5 without affecting rooting rate, and GA with certain concentration is added during proliferation culture ₃ The internode extraction is convenient to operate, and when in secondary proliferation and rooting culture, apple puree with a certain concentration is added, so that seedlings are more robust, leaves are more fresh green, and especially when in rooting culture, the apple puree is matched with PPP ₃₃₃ The root system is made thicker, and finally the survival rate of the hardening seedlings is improved.

Description

Yunnan Hanxiao tissue culture and rapid propagation method and application

Technical field

The invention relates to the technical field of plant tissue culture, specifically a Yunnan Hanxiao tissue culture rapid propagation method and application.

Background technique

Michelia yunnanensis Franch. ex Finet & Gagnep. is a plant of the genus Michelia in the Magnoliaceae family. It is a shrub with dense branches and leaves, up to 4m in height; the buds, twigs, young leaves, petioles and pedicels are densely covered with dark red flat hairs. The leaves are leathery, obovate, narrowly obovate, narrowly obovate-elliptic, 4-10cm long and 1.5-3.5cm wide. The pedicel is thick and short, 3-7mm long, with one bract falling off; the flowers are white and extremely fragrant. Aggregate fruits usually have only 5-9 follicles developed. The follicles are oblate and spherical, 5-8mm wide, with a short tip and residual hairs. There are 1-2 seeds. The flowering period is from March to April and the fruiting period is from August to September.

Distributed in central and southern Yunnan, China. Grows in mountain shrubs at an altitude of 1100-2300m. The flowers are extremely fragrant and the extract can be extracted, making it an excellent ornamental plant; the leaves are fragrant and can be ground into powder to make fragrant noodles. An ancient Yunnan smiling tree found in Lijiang, Yunnan is more than 200 years old. Like the ginkgo tree, it belongs to the Mesozoic era tree species that remained after the Quaternary glaciers and is known as a "living fossil".

At present, the main propagation methods are: sowing and grafting. The survival rate of cutting propagation is low, and graft propagation requires a large number of native trees, which greatly limits the number of grafts. Tissue culture technology is a rapid propagation technology that can quickly produce large quantities of Producing Yunnan smile seedlings is technically difficult, and there is currently no tissue culture and rapid propagation method for Yunnan smile. The existing technology discloses related tissue culture methods such as late spring smiles, Taiwan smiles, Sirindhorn smiles, magnificent smiles, deep mountain smiles, Zhongshan smiles, purple flower smiles, Zuixiang smiles, etc. There are still various problems in the tissue culture process of these methods when applied to Yunnan smiles. This problem is not applicable to the tissue culture of Yunnan smile, so it is urgent to develop a tissue culture method suitable for Yunnan smile.

Contents of the invention

The purpose of the present invention is to provide a Yunnan Xiaoxiao tissue culture rapid propagation method and application in view of the technical problem that the existing technology is not suitable for the Yunnan Xiaoxiao tissue culture culture method.

In order to achieve the above object, the technical solution adopted by the present invention is: a Yunnan smile tissue culture and rapid propagation method, the steps include:

(1) Disinfection of explants: Take the non-lignified stem segments from the end of Yunnan smile, and after cleaning, disinfect them with 0.5% potassium permanganate for 15 to 20 minutes, rinse with sterile water 4 to 5 times, and disinfect with 75% alcohol. 50 to 60 seconds, rinse with sterile water 2 to 3 times; disinfect with 1% potassium permanganate for 5 to 6 minutes, rinse with sterile water for 4 to 5 times, disinfect with 0.05% mercury chloride for 5 to 6 minutes, rinse with sterile water for 5 minutes ~7 times;

(2) First-generation cluster bud induction: sterilized explants are inoculated into cluster bud induction medium and cultured for 20 to 25 days;

The formula of the cluster bud induction medium is: WPM+NAA 0.05~0.08mg/L +2-iP 0.1~0.15mg/L+ZT 2.0~3.0mg/L+activated carbon 0.4~0.6g/L;

(3) Sub-generation proliferation culture: The clustered buds induced in the first generation are inoculated into the sub-generation proliferation medium and cultured for 25 to 35 days;

The formula of the subgeneration proliferation medium is: modified SH medium + IBA 0.1～0.2mg/L + ZT 0.5～0.8mg/L + BR 0.05～0.08mg/L + GA ₃ 0.2～0.3mg/L + apple puree 30～40g/L;

(4) Rooting culture: The value-added buds of the next generation are inoculated into the rooting medium and cultured for 30 to 40 days;

The formula of the rooting medium is: 1/2 improved SH medium + IBA 0.3-0.5 mg/L + IAA 1.5-2.0 mg/L + PPP ₃₃₃ 1.0-2.0 mg/L + applesauce 60-80 g/L + activated carbon 0.2-0.4 g/L;

The modified SH culture medium is: the calcium chloride dosage is changed to 150 mg/L, the EDTA-disodium dosage is changed to 37.3 mg/L, the ferrous sulfate dosage is changed to 27.8 mg/L, and the inositol dosage is changed to 100 mg/L. The remaining ingredients remain unchanged.

Further: the culture conditions of steps (2), (3) and (4) are: temperature 25±2°C, 10-14 hours of daily light, alternating light and dark culture with light intensity of 2000-3000lx.

Further: the formula of the cluster bud induction medium is: WPM+NAA 0.06mg/L +2-iP 0.12mg/L+ZT 2.5mg/L+activated carbon 0.5g/L.

Further: the formula of the subgeneration proliferation medium is: modified SH medium + IBA 0.15 mg/L + ZT 0.7 mg/L + BR 0.06 mg/L + GA ₃ 0.25 mg/L + apple puree 35 g/L.

Further: the formula of the rooting medium is: 1/2 modified SH medium + IBA 0.4mg/L + IAA2.0mg/L + PPP ₃₃₃ 1.5mg/L + apple puree 70g/L + activated carbon 0.3g/L.

The beneficial technical effects of the present invention are:

1. When Yunnan Xiaoxiao established a sterile system, it used conventional 75% alcohol and 0.1% mercuric chloride for disinfection. If the time was too long, the contamination rate could be controlled within a reasonable range. However, 0.1% mercuric chloride explants were harmful to Yunnan Xiaoxiao. The damage is greater, the larger part causes direct death, and the remaining parts are browned, and the browning is extremely serious and leads to the inability to grow. When the time is too short, although the mortality and browning can be greatly reduced, the pollution The rate increased sharply, and sometimes even the entire army was annihilated. When sterilizing the explant, the present invention uses 0.5% potassium permanganate, 75% alcohol, 1% potassium permanganate and 0.05% mercuric chloride solution in order to sterilize. The contamination rate can be controlled within 32%, and the explant will die. The rate is controlled within 5%, and a certain concentration of activated carbon is added to the primary culture medium to control the browning rate within 10%, and neither browning nor activated carbon affects the induction of cluster buds.

2. In the present invention, adding a certain concentration of NAA, 2-iP and ZT to the first generation cluster bud induction medium can increase the induction rate to more than 70%.

3. During the subculture proliferation culture process, high concentrations of 2-iP can cause vitrification. When the concentration is reduced, no matter how the ZT concentration is mixed, it will significantly reduce the proliferation coefficient. In the case of Yunnan Hanxiao In other words, NAA can inhibit the rooting of Yunnan smile. When NAA is used as the subculture proliferation medium for more than 2 generations, the rooting rate drops significantly during rooting culture. At the same time, as the number of subgenerations increases, the rooting rate continues to decrease. In the sub-generation proliferation medium, the present invention changes the NAA of the first generation to a certain concentration of IBA, and at the same time combines a certain concentration of ZT and BR, so that the proliferation coefficient can be stabilized at more than 5, and it does not affect the rooting during subsequent rooting culture. rate, and adding a certain concentration of GA ₃ during proliferation culture can extract the internodes and facilitate the operation during the production process.

4. During the subculture and rooting culture of the present invention, adding a certain concentration of apple puree can make the seedlings more robust and the leaves brighter green. Especially during the rooting culture, the apple puree combined with a certain concentration of PPP ₃₃₃ can make the seedlings more robust. The root system is stronger and stronger, which ultimately improves the survival rate of hardened seedlings.

5. When using WPM, which is commonly used for mother plants, as the basic medium, Yunnan Hanxiao often shows yellowing and falling off of old leaves, and a small amount of tip burning, which seriously affects the proliferation and quality of the seedlings. However, when using SH, it can be obvious Improve but not completely eliminate (as shown in Figure 1). When the concentration of calcium chloride, iron salt and inositol is changed on the basis of SH, the occurrence of yellowing, shedding and burning can be completely eliminated (as shown in Figure 2) shown).

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting any creative effort.

Figure 1 The leaves are bright green when rooting and cultured using modified SH medium;

Figure 2 When the modified SH medium is not used, the leaf sheaths and old leaves turn yellow and fall off;

Figure 3 is a diagram of the first generation induction results in Example 1 of the present invention;

Figure 4 is a diagram of the induction results of the first generation of Comparative Example 8 of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

Example 1

A Yunnan smile tissue culture and rapid propagation method, the steps include:

(1) Explant disinfection: Take the non-lignified stem segment at the end of Michelia yunnanensis, wash it, and then disinfect it with 0.5% potassium permanganate for 15 minutes and rinse it with sterile water for 5 times; disinfect it with 75% alcohol for 50 seconds and rinse it with sterile water for 2 times; disinfect it with 1% potassium permanganate for 5 minutes and rinse it with sterile water for 4 times; disinfect it with 0.05% mercuric chloride for 5 minutes and rinse it with sterile water for 5 times.

(2) First-generation cluster bud induction: The sterilized explants were inoculated into cluster bud induction medium containing WPM+NAA 0.05mg/L +2-iP 0.1mg/L+ZT2.0mg/L+activated carbon 0.4g/L, and placed The cells were cultured at a temperature of 25±2°C, 10 hours of light per day, and alternating light and dark with a light intensity of 2000 lx for 20 days. The results are shown in Figure 3.

(3) Subculture proliferation culture: The clustered buds induced by the first generation were inoculated into modified SH medium + IBA 0.1mg/L + ZT0.5mg/L + BR 0.05mg/L + GA ₃ 0.2mg/L + apple puree 30g/L The cells were placed in a subculture proliferation medium at a temperature of 25 ± 2°C, illuminated for 10 hours a day, and cultured alternately between light and dark at a light intensity of 2000 lx for 25 days.

(4) Rooting culture: The subcultured value-added cluster shoots are inoculated in 1/2 modified SH medium + IBA 0.3mg/L + IAA1.5mg/L + PPP ₃₃₃ 1.0mg/L + apple puree 60g/L + activated carbon 0.2g/L In the rooting medium, place it at a temperature of 25±2°C, illuminate it for 10 hours a day, and cultivate it alternately between light and dark with a light intensity of 2000lx for 30 days.

Among them, the modified SH culture medium is: the calcium chloride dosage is changed to 150 mg/L, the EDTA-disodium dosage is changed to 37.3 mg/L, the ferrous sulfate dosage is changed to 27.8 mg/L, and the inositol dosage is changed to 100 mg/L. The remaining ingredients remain unchanged.

Example 2

A Yunnan smile tissue culture and rapid propagation method, the steps include:

(1) Disinfection of explants: Take the non-lignified stem segments from the end of Yunnan Xiaoxiao. After cleaning, disinfect with 0.5% potassium permanganate for 20 minutes and rinse with sterile water 5 times; disinfect with 75% alcohol for 60 seconds and sterilize Rinse with water 3 times; disinfect with 1% potassium permanganate for 6 minutes, rinse with sterile water 5 times, disinfect with 0.05% mercury chloride for 6 minutes, rinse with sterile water 7 times.

(2) First-generation cluster bud induction: The sterilized explants were inoculated into cluster bud induction medium containing WPM+NAA 0.08mg/L +2-iP 0.15mg/L+ZT 3.0mg/L+activated carbon 0.6g/L, and placed Cultivate at a temperature of 25±2°C, with 14 hours of light every day, and alternating light and dark with a light intensity of 3000lx for 25 days.

(3) Subculture proliferation culture: The clustered buds induced in the first generation were inoculated into modified SH medium + IBA 0.2mg/L + ZT0.8mg/L + BR 0.08mg/L + GA ₃ 0.3mg/L + apple puree 40g/L The cells were cultured in subculture medium at a temperature of 25 ± 2°C, illuminated for 14 hours a day, and alternately light and dark with a light intensity of 3000 lx for 35 days.

(4) Rooting culture: The subcultured value-added cluster shoots are inoculated in 1/2 modified SH medium + IBA 0.5 mg/L + IAA2.0 mg/L + PPP ₃₃₃ 2.0 mg/L + apple puree 80 g/L + activated carbon 0.4 g/L In the rooting medium, place it at a temperature of 25±2℃, illuminate it for 14 hours a day, and cultivate it alternately between light and dark with a light intensity of 3000lx for 40 days.

Among them, the modified SH culture medium is: the calcium chloride dosage is changed to 150 mg/L, the EDTA-disodium dosage is changed to 37.3 mg/L, the ferrous sulfate dosage is changed to 27.8 mg/L, and the inositol dosage is changed to 100 mg/L. The remaining ingredients remain unchanged.

Example 3

A Yunnan smile tissue culture and rapid propagation method, the steps include:

(1) Explant disinfection: Take the non-lignified stem segment from the end of Yunnan Xiaoxiao. After cleaning, disinfect with 0.5% potassium permanganate for 18 minutes and rinse with sterile water 4 times; disinfect with 75% alcohol for 55 seconds and sterilize Rinse with water 3 times; disinfect with 1% potassium permanganate for 5.5 minutes, rinse with sterile water 4 times, disinfect with 0.05% mercury chloride for 5.5 minutes, rinse with sterile water 6 times.

(2) First-generation cluster bud induction: Sterilized explants were inoculated into cluster bud induction medium containing WPM+NAA 0.06mg/L + 2-iP 0.12mg/L+ZT 2.5mg/L+activated carbon 0.5g/L, and placed The cells were cultured at a temperature of 25±2°C, 12 hours of light per day, and alternating light and dark with a light intensity of 2500 lx for 23 days.

(3) Subculture proliferation culture: The clustered buds induced in the first generation were inoculated into modified SH medium + IBA 0.15mg/L + ZT0.7mg/L + BR 0.06mg/L + GA ₃ 0.25mg/L + apple puree 35g/L The cells were cultured in the subculture medium at a temperature of 25±2℃, illuminated for 12 hours a day, and alternately light and dark with a light intensity of 2500 lx for 30 days.

(4) Rooting culture: The subcultured value-added cluster shoots are inoculated in 1/2 modified SH medium + IBA 0.4mg/L + IAA2.0mg/L + PPP ₃₃₃ 1.5mg/L + apple puree 70g/L + activated carbon 0.3g/L In the rooting medium, place it at a temperature of 25±2°C, illuminate it for 12 hours a day, and cultivate it alternately between light and dark with a light intensity of 2500lx for 35 days.

Among them, the modified SH culture medium is: the calcium chloride dosage is changed to 150 mg/L, the EDTA-disodium dosage is changed to 37.3 mg/L, the ferrous sulfate dosage is changed to 27.8 mg/L, and the inositol dosage is changed to 100 mg/L. The remaining ingredients remain unchanged.

Comparative example 1

Take the non-lignified stem segment from the end of Yunnan Hanxia in Example 1, and after cleaning, disinfect Yunnan Hanxia according to the method disclosed in Example 1 of the patent "Tissue Culture Propagation Method of Late Spring Hanxia" (publication number: CN100429972 A), and then perform Induced differentiation culture, value-added culture for successive generations, and rooting culture.

Comparative example 2

Take the non-lignified stem segment from the end of Yunnan Hanxiao in Example 1, and after cleaning, perform tissue culture on Yunnan Hanxiao according to the method embodiment disclosed in the patent "A Taiwanese Hanxiao Tissue Culture Propagation Method" (publication number: CN103392601A). Clustered buds can be induced, and subsequent proliferation and rooting culture are not carried out.

Comparative example 3

Take the non-lignified stem segments at the end of Yunnan Hanxiao in Example 1. After cleaning, use the method disclosed in the patent "A Method for Regenerating Plants of Sirindhorn Hanxiao in vitro Culture" (publication number: CN 103718969 A) to treat Yunnan. Tissue culture was performed with smile.

Comparative example 4

Take the non-lignified stem segment at the end of Yunnan Smile in Example 1, perform explant disinfection and primary induction culture according to the method of Example 1 of the present invention, and then follow the patent "A Method for Aseptic Germination and Rapid Propagation of Magnificent Smile Seeds" ( Publication number: CN112106664 A) Disclosed method example 1: Tissue culture of Yunnan smile.

Comparative example 5

Take the non-lignified stem segment at the end of Yunnan Hanxiao in Example 1. After cleaning, follow Qiu Jian's "Research on Cutting Propagation and Tissue Culture Technology of Zhongshan Hanxiao" [D]. Nanjing Forestry University, 2023. DOI: 10.27242/d.cnki. The best method disclosed by gnjlu.2023.000286 is used to carry out explant disinfection, primary induction culture, secondary proliferation culture and rooting culture of Yunnan smile.

Comparative example 6

Take the non-lignified stem segment at the end of Yunnan Hanxiao in Example 1, perform explant disinfection and primary induction culture according to Example 1, and then follow Cheng Qiangqiang's "Establishment of Tissue Culture and Rapid Propagation System of Purple Flower Hanxiao" [J]. Journal of Forestry Engineering, 2014, 28(01):118-121.DOI:10.13360/j.issn.1000-8101.2014.01.031. The best disclosed method for subculture and rooting culture of Yunnan smile.

Comparative example 7

Take the unlignified stem segment at the end of Yunnan Hanxiao in Example 1 and follow the "Tissue Culture and Plant Regeneration of Zuixiang Hanxiao" published by Li Xue [J]. Plant Physiology Communications, 2005, 41(6): 1. DOI: CNKI :SUN:ZWSL.0.2005-06-027. The best disclosed method is to carry out explant disinfection, primary induction culture, secondary proliferation culture and rooting culture of Yunnan Hanxiao.

Comparative example 8

According to the best method of Du Ting et al. "Research on rapid propagation technology and callus induction of Yunnan smile tissue culture." Journal of Yunnan Agricultural University: Natural Science Edition (2013). Explant disinfection, primary induction culture, and Subculture was propagated and cultured, and rooting culture was performed according to Example 1 of the present invention. The results are shown in Figure 4.

Table 1 Comparison results

Disinfection contamination rate Disinfection mortality rate Browning rate Cluster bud induction rate proliferation coefficient Rooting rate Rooted seedling state Seedling survival rate Example 1 22.6% 12.2% 5.3% 76.8% 5.5 81.6% robust 96.7% Example 2 20.1% 12.7% 3.9% 82.6% 6.1 82.9% robust 97.3% Example 3 28.8% 13.6% 7.2% 84.7% 6.9 80.2% robust 95.2% Comparative example 1 21.3% 46.4% 69.0% 21.3% 2.1 41.2% weaker 90.6% Comparative example 2 17.1% 49.3% 72.6% 0 —— —— —— —— Comparative example 3 79.7% 10.3% 16.9% 19.6% 1.7 19.2% weaker 87.6% Comparative example 4 28.6% 13.2% 4.1% 74.6% 2.9 40.6% Stronger 97.4% Comparative example 5 74.9% 0 3.6% 19.4% 1.2 3.9% Germination is difficult 94.3% Comparative example 6 23.1% 11.6% 5.4% 81.4% 3.4 34.2% weaker 84.2% Comparative example 7 10.4% 71.3% 83.0% 21.7% 2.6 35.7% weaker 92.9% Comparative example 8 16.6% 54.9% 70.8% 24.4% 3.1 70.8% Stronger 98.6%

It can be seen from Table 1 that in Examples 1-3 of the present application, most indicators are significantly better than Comparative Examples 1-8. Although some indicators are slightly lower than some indicators in Comparative Examples, the comprehensive indicators are obviously better than Comparative Examples 1-8. 8.

Finally, it should be noted that the above embodiments are only used to illustrate but not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the present invention can still be modified. Or equivalent substitutions, and any modification or partial substitution without departing from the spirit and scope of the present invention shall be covered by the scope of the claims of the present invention.

Claims (6)

1. The tissue culture and rapid propagation method for michelia yunnanensis is characterized by comprising the following steps of:

(1) Explant sterilization: taking the stem sections of michelia yunnanensis which are not lignified at the end parts, cleaning, and then sequentially disinfecting with 0.5% potassium permanganate for 15-20 min and washing with sterile water for 4-5 times; sterilizing with 75% alcohol for 50-60 s, and rinsing with sterile water for 2-3 times; sterilizing for 5-6 min by 1% potassium permanganate, rinsing for 4-5 times by sterile water, sterilizing for 5-6 min by 0.05% mercuric chloride, and rinsing for 5-7 times by sterile water;

(2) And (3) primary cluster bud induction: inoculating the sterilized explant into a cluster bud induction culture medium for culturing for 20-25 days;

the cluster bud induction culture medium comprises the following formula: WPM+NAA 0.05-0.08 mg/L+2-iP 0.1-0.15 mg/L+ZT 2.0-3.0 mg/L+activated carbon 0.4-0.6 g/L;

(3) And (3) subculturing and proliferation: inoculating cluster buds induced by the first generation into a secondary proliferation culture medium for culturing for 25-35 days;

the formula of the secondary proliferation culture medium is as follows: improved SH culture medium, IBA 0.1-0.2 mg/L, ZT 0.5-0.8 mg/L, BR 0.05-0.08 mg/L and GA ₃ 0.2-0.3 mg/L and 30-40 g/L of apple puree;

(4) Rooting culture: inoculating the cluster buds with the secondary multiplication value into a rooting culture medium for culturing for 30-40 days;

the rooting culture medium comprises the following formula: 1/2 modified SH culture medium+IBA 0.3-0.5 mg/L+IAA 1.5-2.0 mg/L+PPP ₃₃₃ 1.0 to 2.0mg/L, 60 to 80g/L of apple puree and 0.2 to 0.4g/L of activated carbon;

the improved SH culture medium is as follows: the dosage of calcium chloride is changed to 150mg/L, the dosage of EDTA-disodium is changed to 37.3mg/L, the dosage of ferrous sulfate is changed to 27.8mg/L, the dosage of inositol is changed to 100mg/L, and the rest components are unchanged.

2. The method according to claim 1, wherein: the culture conditions of steps (2), (3) and (4) are: the culture medium is alternately cultured at the temperature of 25+/-2 ℃ under the light intensity of 2000-3000 lx and the light irradiation is carried out for 10-14 hours every day.

3. The method according to claim 1, wherein: the cluster bud induction culture medium comprises the following formula: WPM+NAA 0.06 mg/L+2-iP 0.12 mg/L+ZT2.5 mg/L+activated carbon 0.5g/L.

4. The method according to claim 1, wherein: the formula of the secondary proliferation culture medium is as follows: improved SH culture medium, IBA 0.15mg/L, ZT 0.7mg/L, BR 0.06mg/L and GA ₃ 0.25 mg/L+35 g/L apple puree.

5. The method according to claim 1, wherein: the rooting culture medium comprises the following formula: 1/2 modified SH Medium+IBA 0.4mg/L+IAA 2.0mg/L+PPP ₃₃₃ 1.5mg/L, 70g/L of apple puree and 0.3g/L of activated carbon.

6. Use of the method according to any one of claims 1-5 in tissue culture and rapid propagation of michelia yunnanensis.