WO2022171212A2 - Method for ex vivo culturing of thornless green prickly ash zanthoxylum armatum dc - Google Patents

Abstract

Provided in the present invention is a method for the ex vivo culturing of thornless green prickly ash Zanthoxylum armatum DC, belonging to the technical field of plant tissue culture. According to the present invention, a non-axillary bud stem section of thornless green prickly ash Zanthoxylum armatum DC is used as an explant. By means of reasonably formulating a callus induction medium, the non-axillary bud stem section is successfully induced to produce callus, so as to produce an adventitious bud for propagation, thereby filling the technical gap of using a non-axillary bud stem section of thornless green prickly ash Zanthoxylum armatum DC as an explant for regeneration of plants by means of callus. According to the present invention, a propagation system for the ex vivo regeneration of a non-axillary bud stem section of thornless green prickly ash Zanthoxylum armatum DC is established for the first time, and large-scale production is realized.

Description

A kind of in vitro culture method of thornless Chinese prickly ash technical field

The invention belongs to the technical field of plant tissue culture, and in particular relates to an in vitro culture method of prickly pear.

Background technique

Thornless green prickly ash Zanthoxylum armatum DC is a natural bud variety of green pepper (Zanthoxylum armatum DC, with thorns), which has the characteristics of no thorns (less thorns). Rongchang thornless prickly ash has been approved as an improved tree species in Chongqing (the improved variety number: Yu S-SV-ZA-001-2016). Canghuajiao is different from the thornless red pepper varieties (Zanthoxylum bungeanum Maxim) cultivated by my country itself.

Under normal circumstances, the propagation method of common green pepper is seed propagation. Since the thornless Chinese prickly ash is a natural bud, its character is unstable. If it is propagated by seed sowing, its thornless character will disappear, and it will become a thorny green prickly ash again. We call this phenomenon an atavistic phenomenon. Therefore, the propagation of budding varieties usually cannot be carried out by seed sowing, and asexual propagation methods such as cuttings, grafting, and in vitro culture are often used. Compared with traditional vegetative propagation such as cuttings (limited cuttings, low rooting rate), grafting (limited by the number of scions and seasons, and high labor costs), the in vitro culture vegetative propagation technology is not limited by the number of spikes, and has the advantages of annual production, The advantages of factory production, fast reproduction, high efficiency and good uniformity can better meet the large-scale demand of the market.

Disclosed in the prior art is a method of culturing the stem section with axillary buds of Chinese prickly ash without thorns as explants, and propagating by the mode of budding (referring to IN VITRO PROPAGATION METHOD OF TISSUE CULTURE SEEDLINGS OF ZANTHOXYLUM ARMATUM, PCT patent; Authorized country: Netherlands; Patent number: 2027681). However, this method has limitations in the selection of explants, and requires that the stem segments used as explants need to have axillary buds.

At present, there is no in vitro culture method using the stem segments without axillary buds of Chinese prickly ash as explants.

SUMMARY OF THE INVENTION

In view of this, the object of the present invention is to provide a kind of in vitro culture method of Chinese prickly ash without thorns, the present invention can take the axillary budless stem section of prickly pear without thorns as explants, by inducing callus and then produce adventitious buds for propagation , which fills the technical gap of obtaining regenerated plants through callus by using the stem segments without axillary buds of Chinese prickly ash as explants.

The invention provides a kind of in vitro culture method of thornless Chinese prickly ash, comprising the following steps:

1) inoculate the stem section without axillary bud of Chinese prickly ash without a thorn in a callus induction medium, carry out induction culture, and grow callus to the stem section;

2) inoculate the stem segment of the callus with the differentiation medium, carry out differentiation culture, and obtain clumps of buds;

3) inoculating the clustered buds in a proliferation medium, carrying out proliferation culture to obtain sprouts;

4) inoculating the sprouts in a rooting medium, carrying out rooting culture to obtain rooting seedlings;

The callus induction medium is based on MS, and includes components with the following concentrations: ZR 1.3-1.7 mg/L, agar 5.8-6.2 g/L, sucrose 29.8-30.2 g/L and the volume percentage of 0.1% PPM.

Preferably, the differentiation medium is MS-based medium, and includes components with the following concentrations: ZR 1.8-2.2 mg/L, agar 5.8-6.2 g/L, sucrose 29.8-30.2 g/L and volume percentages is 0.1% PPM.

Preferably, the proliferation medium is based on MS, and includes components with the following concentrations: ZR 1-1.5 mg/L, IBA 0.08-0.12 mg/L, agar 5.8-6.2 g/L and sucrose 29.8-29.8 mg/L 30.2g/L.

Preferably, the rooting medium is based on 1/2 MS, and includes components with the following concentrations: IBA 0.1-0.15 mg/L, agar 5.8-6.2 g/L and sucrose 29.8-30.2 g/L.

Preferably, the induction culture includes culturing in the dark for 2-3 days and then culturing in the light for 20-25 days, and the light intensity of the light-culturing is 800-1000 lux.

Preferably, the differentiation culture is carried out under light conditions, and the light conditions for the differentiation culture include: the light intensity is 1800-2000 lux; the time of the differentiation and culture is 25-30 d.

Preferably, the proliferation culture includes culturing in the dark for 8-9 days and then performing light culture, until the stem segment grows to a height of 3-4 cm and grows 3-4 internodes; The first light culture and the second light culture; the light intensity of the first light culture is 800-1000 lux; the time of the first light culture is 7-8 d; the light intensity of the second light culture is 1800-2000 lux.

Preferably, the rooting culture includes culturing in a dark environment for 7-9 days and then performing light culture for 7-21 days; the light intensity of the light culture is 1800-2000 lux.

Preferably, after obtaining the rooted seedlings, the method further includes performing hardening and domestication transplanting on the rooted seedlings to obtain tissue cultured seedlings.

Preferably, the seedling hardening is carried out in a greenhouse with a shading rate of 65%-80%, the seedling hardening time is 8-12 d, and the temperature of the seedling hardening is 20-30°C.

The invention provides an in vitro culture method of prickly ashless prickly ash. The invention uses the axillary budless stem section of prickly ashless prickly ash as an explant, and through rational preparation of a callus induction medium, the axillary budless stem section is successfully induced to produce callus Then, adventitious buds are produced for propagation, which fills the technical gap of plant regeneration through callus by using the stem segments without axillary buds of Chinese prickly ash as explants. The invention establishes for the first time a propagation system for the in vitro regeneration of the non-thornless Chinese prickly ash without axillary bud stems, and realizes large-scale production.

The stem segment with axillary buds is obtained by inducing the germination and elongation of axillary buds, without de-differentiation and re-differentiation. and re-differentiation stage, in order to regenerate adventitious buds to form sprouts, the induction process is complicated. The invention uses the axillary budless stem section of thornless prickly ash as the explant and has the following advantages: the axillary budless stem section is more widely used as a raw material; the pollution point of the stem section culture mainly comes from the axillary bud, and the axillary budless stem section is the explant, The contamination rate is greatly reduced, and the acquisition rate is higher; the buds obtained from the stem segments with axillary buds are single buds obtained from axillary buds; the buds obtained from the stem segments without axillary buds are bud clusters obtained from callus (multiple buds). ), the method of the present invention can obtain clumps of sprouts, and realizes the rapid propagation of thornless prickly ash seedlings.

Description of drawings

Fig. 1 is a branch and a leaf of Chinese prickly ash without thorns;

Figure 2 is the back of a thornless prickly ash leaf;

Fig. 3 is that the stem segment without axillary bud is an explant;

Fig. 4 is that the stem segment without axillary buds produces callus and differentiates adventitious buds;

Fig. 5 is that adventitious bud forms bud cluster;

Fig. 6 is subculture of clump bud;

Fig. 7 is the elongation growth of clump bud;

Fig. 8 is the cutting and rooting of clump buds;

Figure 9 is a greenhouse substrate seedling;

Figure 10 is a commercial seedling.

Detailed ways

The invention provides a kind of in vitro culture method of thornless Chinese prickly ash, comprising the following steps:

1) inoculate the stem section without axillary bud of Chinese prickly ash without a thorn in a callus induction medium, carry out induction culture, and grow callus to the stem section;

2) inoculate the stem segment of the callus with the differentiation medium, carry out differentiation culture, and obtain clumps of buds;

3) inoculating the clustered buds in a proliferation medium, carrying out proliferation culture to obtain sprouts;

4) inoculating the sprouts in a rooting medium, carrying out rooting culture to obtain rooting seedlings;

The callus induction medium is based on MS, and includes components with the following concentrations: ZR 1.3-1.7 mg/L, agar 5.8-6.2 g/L, sucrose 29.8-30.2 g/L and the volume percentage of 0.1% PPM.

In the present invention, firstly, the stem segments without axillary buds of Chinese prickly ash are inoculated into a callus induction medium, and induction culture is carried out until callus grows out of the stem segments; the callus induction medium is based on MS, including the following Concentration components: ZR 1.3～1.7mg/L, agar 5.8～6.2g/L, sucrose 29.8～30.2g/L and PPM with a volume percentage of 0.1%. In the present invention, the thornless Chinese prickly ash is preferably Rongchang thornless prickly ash.

In the present invention, the callus induction medium preferably includes the following components in the concentration: ZR 1.5mg/L, agar 6g/L, sucrose 30g/L and PPM with a volume percentage of 0.1%.

In the present invention, the stem section without axillary buds of the thornless Chinese prickly ash is preferably taken from the annual branch of the thornless prickly ash; the stem section is preferably a semi-lignified stem section; The length of the segment is preferably 0.5 to 1 cm. In the present invention, before inoculating the axillary budless stem section of Chinese prickly ash without callus inducing medium, it is preferable to sterilize the stem section. Conventional explant disinfection methods in the field are sufficient.

In the present invention, the induction culture preferably includes culturing in the dark for 2-3 d and then culturing in the light for 20-25 d, and the light intensity of the light-culturing is preferably 800-1000 lux. In the present invention, the stem segment grows callus through the induction culture, and a small amount of callus further forms adventitious buds at this stage. The adventitious buds will continue to elongate in the differentiation medium, and like the adventitious buds obtained by callus differentiation, they can become the subsequent clump buds, which are then cut into rooted shoots.

After the callus grows out of the stem segment, the present invention inoculates the stem segment from which the callus grows into a differentiation medium, and performs differentiation culture to obtain cluster buds.

In the present invention, the differentiation medium is MS-based medium, and preferably includes the following components in concentrations: ZR 1.8-2.2 mg/L, agar 5.8-6.2 g/L, sucrose 29.8-30.2 g/L and The PPM with a volume percentage of 0.1% more preferably includes the following concentrations of components: ZR 2mg/L, agar 6g/L, sucrose 30g/L and PPM with a volume percentage of 0.1%.

In the present invention, the differentiation culture is preferably carried out under light conditions, and the light conditions for the differentiation culture preferably include: the light intensity is 1800-2000 lux; the time of the differentiation culture is preferably 25-30 d. The present invention obtains clump buds through the differentiated culture, and can improve the efficiency of plant tissue culture. In the present invention, the clump buds grow on the stem segments, and further comprises cutting the clump buds from the stem segments, and this step is performed in a sterile environment by using sterilized tools.

After the clump buds are obtained, the present invention inoculates the clump buds in a proliferation medium, and performs proliferation culture to obtain sprouts.

In the present invention, the proliferation medium is MS-based medium, and preferably includes components with the following concentrations: ZR 1-1.5 mg/L, IBA 0.08-0.12 mg/L, agar 5.8-6.2 g/L and Sucrose 29.8～30.2g/L, more preferably, the following components are included in the concentration: ZR 1～1.5mg/L, IBA 0.1mg/L, agar 6g/L and sucrose 30g/L.

In the present invention, the proliferation culture includes culturing in the dark for 8-9 days and then performing light culture, until the stem segment grows to a height of 3-4 cm and grows 3-4 internodes; the light culture includes sequentially performing The first light culture and the second light culture; the light intensity of the first light culture is 800～1000lux; the time of the first light culture is 7～8d; the light intensity of the second light culture is 1800～1000lux 2000lux. In the present invention, the time of the second illumination cultivation is preferably 13-14 d. The invention is beneficial to the smooth transition of the stem segment from the dark environment to the light environment by gradually increasing the light intensity. More conducive to stem growth.

After the sprouts are obtained, the present invention inoculates the sprouts in a rooting medium, and performs rooting culture to obtain rooted seedlings.

In the present invention, the sprout preferably has a height of ≥ 2.0 cm and a stem diameter of ≥ 2.0 mm; the depth of the sprout inserted into the rooting medium is preferably 1.5-2 cm.

In the present invention, the rooting medium is based on 1/2 MS, and preferably includes the following components: IBA 0.1-0.15 mg/L, agar 5.8-6.2 g/L and sucrose 29.8-30.2 g/L L, more preferably include the following concentrations of components: IBA 0.1 ~ 0.15mg/L, agar 6g/L and sucrose 30g/L.

In the present invention, the rooting culture includes culturing in a dark environment for 7-9 days and then performing light culture for 7-21 days; the light intensity of the light culture is preferably 1800-2000 lux; The number of root systems is ≥2 and the root length is ≥2.0cm.

In the present invention, the culture in the dark environment is preferably cultured under the condition that the lights and curtains are turned off in the culture room, and the culture flask is covered with a black film at the same time.

In the present invention, from the stage of induction culture to rooting culture, if light culture is required, the daily light culture time is preferably 10h; from the stage of induction culture to rooting culture, the culture temperature is preferably 23-25°C.

After the rooted seedlings are obtained, the present invention preferably further includes performing hardening and domestication transplanting on the rooted seedlings to obtain tissue cultured seedlings.

In the present invention, the seedling hardening is preferably carried out in a greenhouse with a shading rate of 65% to 80%. During the seedling hardening process, the thornless Chinese prickly ash bottle seedlings are placed in the greenhouse, and the cap of the culture bottle is loosened. The time of the hardening is preferably 8-12d, more preferably 10d; the temperature of the hardening is preferably 20-30°C; the role of the hardening is to thicken and thicken the leaves of the rooted seedlings.

After the hardening of the seedlings, the present invention also includes rinsing the rooting medium on the rooting seedlings and soaking the whole plant in a sterilizing solution; the reagent used for the rinsing is preferably clear water; the sterilizing solution is preferably 1000 times the carbendazim solution; the soaking time is preferably 1-2min.

After the soaking, the present invention transplants the rooted seedlings soaked in the sterilizing liquid into a matrix plug tray for domestication; the matrix includes the following raw materials by volume: 7 parts of peat and 3 parts of perlite.

After the transplanting, the present invention preferably further includes watering the transplanted seedlings with root-fixing water, and then covering with a film to keep warm and moisturizing.

In the present invention, the domestication is preferably carried out in a greenhouse; morning and evening ventilation is performed during the domestication process; compound fertilizer is sprayed on the 11th day of the domestication; N:P:K=15 of the compound fertilizer : 15:15; the mass concentration of the compound fertilizer is preferably 0.5%; the application frequency of the compound fertilizer is preferably once every 7d; the 15th day of the domestication removes the film, and continues to cultivate, after 30 to 45 days out of the nursery.

Unless otherwise specified, the present invention has no special requirements on the source of the raw materials used, and commercially available commodities well known to those skilled in the art can be used.

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Example 1

S1. Disinfection of explants: take the current-year branches of Chinese prickly ash without tattoos (Figures 1-2) as explants, take the semi-lignified stems without axillary buds after disinfection and place them in the sterile inoculation tray on the ultra-clean workbench. Cut the stem section of prickly ash without thorns into 0.5-1.0 cm stem sections (without leaves and axillary buds), as shown in Figure 3.

S2. Callus induction: Place the cut stem segments in S1 in a callus induction medium, and after culturing in a dark environment for 2 to 3 days, place the light intensity at 1000 lux for 20 to 25 days to obtain callus and a small amount of adventitious buds, at this stage, callus was formed, and a small amount of callus further formed adventitious buds, as shown in Figure 4.

The formula of callus induction medium is: MS+ ^ZR1.5mg L-1+agar ^6.0g L ^-1 +sucrose 30g L-1+0.1% PPM by volume;

S3. Differentiation culture: The callus-grown stem segments are placed flat in the differentiation medium, and placed in a light intensity of 2000 lux to continue culturing for 25-30 d, until cluster buds are obtained, as shown in Figure 5 .

The formula of the differentiation medium is: MS+ZR 2.0 mg L ^-1 + agar 6.0 g L ^-1 + sucrose 30 g L ^-1 + 0.1% PPM by volume.

S4, cutting and subculture: Place the Zanthoxylum bungeanum stem segment that has grown cluster buds in a sterile inoculation tray on an ultra-clean workbench, and cut off the tissue growing cluster buds with sterilized tools, as shown in Figure 6 .

S5. Subsequent proliferation: put the clustered buds cut in S4 into the proliferation medium, and after culturing in the dark environment for 8 to 9 days, place them in time under the condition of light intensity of 1000 lux for 7 to 8 days, and then increase the light intensity Increase to 000 lux and cultivate for 13-14 days, until the stem segment grows to a height of 3-4 cm and has 3-4 internodes, as shown in Figure 7.

The formula of the proliferation medium is: MS+ZR 1.0 mg L ^-1 + IBA 0.1 mg·L ^-1 + agar 6.0 g L ^-1 + sucrose 30 g L ^-1 .

During the stage of S1-S5, in the process of light culture, the daily light culture time was 10h, and the temperature was 23-25°C.

S6. Rooting culture: The sprouts obtained by subculture are cut and inoculated into the rooting medium. The sprouts are ≥2.0cm high, the stem diameters are ≥2.0mm, and the depth of insertion into the medium is 1.5-2.0cm; The environment was cultured for 7-9 days, and then transferred to the light environment for 7-14 days. The light culture time was 10h/d. During the whole rooting cultivation process, the temperature was 23-25 °C, and the light intensity was 2000 lux; after culturing for 14-21 d, the rooting seedlings with ≥2 root systems and root length ≥2.0 cm were selected for transplantation, as shown in Figure 8.

Wherein, the medium composition in the rooting culture process is as follows: 1/2MS basal medium, the dosage of IBA is 0.1 mg L ^-1 , the dosage of agar is 6.0 g L ^-1 , and the dosage of sucrose is 30 g L ^-1 .

S7, domestication and transplanting: first place the thornless prickly ash bottle seedlings in a greenhouse with a shading rate of 65% to 80%, loosen the cap of the culture bottle, and refine the seedlings at room temperature for about 10 days to thicken and thicken the leaves of the rooted seedlings. Then rinse the rooting seedling medium after the seedling hardening with clean water, then soak it in 1000 times carbendazim solution for 1-2 minutes, and plant it in the prepared matrix plug tray. The proportion of transplanting matrix is peat: perlite. (V:V=7:3), after planting, irrigate root-fixing water, cover with film to keep warm and moisturizing, place in greenhouse for cultivation, pay attention to ventilation in the morning and evening during transplanting and cultivation, and apply 0.5% compound fertilizer (N:P :K=15:15:15) spray, once every 7d, remove the film after about 15d, and can be out of the garden after 30-45d, as shown in Figure 9 and Figure 10.

Example 2

S1. Disinfection of explants: take the current year's branches of Chinese prickly ash as the explants, take the semi-lignified stems without axillary buds after disinfection and place them in the sterile inoculation tray on the ultra-clean workbench, and cut the stems of the non-thorned Chinese prickly ash into a sterile inoculation tray. Stem segments of 0.5-1.0 cm (without leaves and axillary buds).

S2. Callus induction: Place the cut stem segments in S1 in a callus induction medium, and after culturing in a dark environment for 2 to 3 days, place the light intensity at 1000 lux for 20 to 25 days to obtain callus and a small amount of adventitious buds, at this stage, callus is formed, and a small amount of callus further forms adventitious buds.

The formula of callus induction medium is: MS+ ^ZR1.5mg L-1+agar ^6.0g L ^-1 +sucrose 30g L-1+0.1% PPM by volume;

S3. Differentiation culture: The callus-grown stem segments are placed flat in the differentiation medium, and placed in a light intensity of 2000 lux to continue culturing for 25-30 days, until cluster buds are obtained.

The formula of the differentiation medium is: MS+ZR 2.0 mg L ^-1 + agar 6.0 g L ^-1 + sucrose 30 g L ^-1 + 0.1% PPM by volume.

S4, cutting and sub-generation: place the stem section of Zanthoxylum bungeanum with clumps of buds in a sterile inoculation tray on the ultra-clean workbench, and cut off the tissue with clumps of buds with sterilized tools.

S5. Subsequent proliferation: put the clustered buds cut in S4 into the proliferation medium, and after culturing in the dark environment for 8 to 9 days, place them in time under the condition of light intensity of 1000 lux for 7 to 8 days, and then increase the light intensity Increase to 000 lux and cultivate for 13-14 days, until the stem segment grows to a height of 3-4 cm and has 3-4 internodes, as shown in Figure 7.

The formula of the proliferation medium is: MS+ZR 1.5 mg L ^-1 + IBA 0.1 mg·L ^-1 + agar 6.0 g L ^-1 + sucrose 30 g L ^-1 .

During the stage of S1-S5, in the process of light culture, the daily light culture time was 10h, and the temperature was 23-25°C.

S6. Rooting culture: The sprouts obtained by subculture are cut and inoculated into the rooting medium. The sprouts are ≥2.0cm high, the stem diameters are ≥2.0mm, and the depth of insertion into the medium is 1.5-2.0cm; The environment was cultivated for 7-9 days, and then transferred to the light environment to continue cultivation. The light culture time was 10h/d. During the whole rooting cultivation process, the temperature was 23-25 °C, and the light intensity was 2000 lux; after culturing for 14-21 d, the rooting seedlings with the number of root systems ≥ 2 and the root length ≥ 2.0 cm were selected for transplantation.

Wherein, the medium composition in the rooting culture process is as follows: 1/2MS basal medium, the dosage of IBA is 0.15 mg L ^-1 , the dosage of agar is 6.0 g L ^-1 , and the dosage of sucrose is 30 g L ^-1 .

S7, domestication and transplanting: first place the thornless prickly ash bottle seedlings in a greenhouse with a shading rate of 65% to 80%, loosen the cap of the culture bottle, and refine the seedlings at room temperature for about 10 days to thicken and thicken the leaves of the rooted seedlings. Then rinse the rooting seedling medium after the seedling hardening with clean water, then soak it in 1000 times carbendazim solution for 2 minutes, and plant it in the prepared matrix plug tray. The ratio of transplanting matrix is peat: perlite (V :V=7:3), after planting, irrigate root-fixing water, cover with film to keep warm and moisturizing, place it in a greenhouse for cultivation, pay attention to ventilation in the morning and evening during the transplanting and cultivation process, and use 0.5% compound fertilizer (N:P:K) after 10 days. =15:15:15) spray, once every 7d, remove the film after about 15d, and can be out of the nursery after 30-45d.

Although the above embodiment has made a detailed description of the present invention, it is only a part of the embodiments of the present invention rather than all of the embodiments, and people can also obtain other embodiments according to the present embodiment without creativity, and these embodiments are all It belongs to the protection scope of the present invention.

Claims (10)

1. A method for culturing in vitro without thorns prickly ash, comprising the following steps:

   1) inoculate the stem section without axillary bud of Chinese prickly ash without a thorn in a callus induction medium, carry out induction culture, and grow callus to the stem section;

   2) inoculate the stem segment of the callus with the differentiation medium, carry out differentiation culture, and obtain clumps of buds;

   3) inoculating the clustered buds in a proliferation medium, carrying out proliferation culture to obtain sprouts;

   4) inoculating the sprouts in a rooting medium, carrying out rooting culture to obtain rooting seedlings;

   The callus induction medium is based on MS, and includes components with the following concentrations: ZR 1.3-1.7 mg/L, agar 5.8-6.2 g/L, sucrose 29.8-30.2 g/L and the volume percentage of 0.1% PPM.
2. The in vitro culturing method according to claim 1, wherein the differentiation medium is MS-based medium, and comprises the following components: ZR 1.8-2.2 mg/L, agar 5.8-6.2 g/L , 29.8-30.2g/L of sucrose and PPM with a volume percentage of 0.1%.
3. The in vitro culturing method according to claim 1, wherein the proliferation medium is based on MS, and comprises the following concentrations of components: ZR 1-1.5 mg/L, IBA 0.08-0.12 mg/L , agar 5.8～6.2g/L and sucrose 29.8～30.2g/L.
4. The in vitro culturing method according to claim 1, wherein the rooting medium takes 1/2MS as a basal medium, and comprises the following concentrations of components: IBA 0.1-0.15 mg/L, agar 5.8-6.2 g /L and sucrose 29.8～30.2g/L.
5. The in vitro culturing method according to claim 1, wherein the induction culturing comprises culturing in the dark for 2-3 d and then cultivating with light for 20-25 d, and the light intensity of the light-culturing is 800-1000 lux.
6. The in vitro culturing method according to claim 1, wherein the differentiation culture is carried out under light conditions, and the light conditions for the differentiation culture include: the light intensity is 1800-2000 lux; the time of the differentiation culture is 25 ~30d.
7. The in vitro culturing method according to claim 1, wherein the proliferation culture comprises culturing under dark conditions for 8-9 days and then performing light culture, until the stem segment grows to a height of 3-4 cm and grows 3-4 cm. an interval;

   The illumination culture includes a first illumination culture and a second illumination culture performed in sequence; the illumination intensity of the first illumination culture is 800-1000 lux; the time of the first illumination culture is 7-8 days; the second illumination The light intensity of the culture was 1800-2000 lux.
8. The in vitro culturing method according to claim 1, wherein the rooting culture comprises culturing in a dark environment for 7-9 days and then culturing in a dark environment for 7-21 d; the illumination intensity of the culturing in light is 1800-2000 lux.
9. The in vitro culturing method according to claim 1, characterized in that, after obtaining the rooted seedlings, the method further comprises carrying out hardening and domestication transplanting on the rooted seedlings to obtain tissue cultured seedlings.
10. The in vitro culturing method according to claim 9, wherein the seedling hardening is carried out in a greenhouse with a shading rate of 65% to 80%, and the time for the seedling hardening is 8 to 12 days; The temperature is 20 ~ 30 ℃.

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