Tissue culture method of machilus brevisepala

Abstract

The application provides a tissue culture method of Acronychia fruticosa. The method comprises the following steps: selecting semi-lignified Acronychia fruticosa sprout strips as explants; sequentially performing disinfection treatment, primary induction culture, first proliferation culture, disinfection treatment and rooting culture on the explants; the disinfection treatment comprises sequentially performing seedling strengthening culture and second proliferation culture; the culture medium of the first proliferation culture and the second proliferation culture both comprises a first basic culture medium, and further comprises 0.4-0.6 mg / L 6-BA, 14-16 g / L sucrose and 5-7 g / L carrageenan based on the first basic culture medium; and the first basic culture medium is WPM. The application improves the culture medium formula by exploring the influence of different basic culture media, the concentration of 6-BA and the concentration of sucrose on proliferation and rooting, so that the proliferation rate of the tissue culture of Acronychia fruticosa reaches more than 2.74, and the rooting rate reaches more than 95%.

Description

Technical Field

[0001] This application relates to the field of plant propagation technology, and in particular to a method for tissue culture of Conophytum purpureus. Background Technology

[0002] The red pine (Castanopsis hystrix) is an evergreen tree belonging to the genus Castanopsis in the family Fagaceae. It is the fastest-growing precious tree species with the largest planting area in southern my country. Its wood is hard and has a large proportion of heartwood, which can be used to cultivate large-diameter precious timber. In 2017, it was included in the "Reference List of Major Cultivated Precious Tree Species in China" by the National Forestry and Grassland Administration.

[0003] As the area under cultivation for Castanopsis fargesii increases year by year, the imbalance between supply and demand for high-quality seedlings has become increasingly prominent. Common propagation methods for Castanopsis fargesii include seed propagation, grafting, and cuttings. However, the limited number of seeds and cuttings severely affects the large-scale propagation and widespread application of high-quality seedlings.

[0004] Therefore, there is an urgent need to improve the traditional propagation methods of the red cone. Summary of the Invention

[0005] Based on this, one or more embodiments of this application provide a tissue culture method for Conophytum purpureus that has both a high proliferation rate and a high rooting rate.

[0006] According to a first aspect of the embodiments of this application, a method for tissue culture of *Pygium wilfordii* is provided, comprising the following steps:

[0007] Semi-lignified red cone scions were selected as explants; and

[0008] The explants were subjected to disinfection, primary induction culture, first proliferation culture, sterilization treatment, and rooting culture in sequence.

[0009] The sterilization treatment includes sequential seedling cultivation and a second proliferation culture;

[0010] The culture media for the first and second proliferation cultures include a first basal medium, and based on the first basal medium, it also contains 0.4 mg / L to 0.6 mg / L 6-BA, 14 g / L to 16 g / L sucrose and 5 g / L to 7 g / L carrageenan; the first basal medium is selected from WPM.

[0011] In one embodiment, the culture medium for the primary induction culture includes a second basal medium, and based on the second basal medium, it further contains 1.8 mg / L to 2.2 mg / L 6-BA, 0.04 g / L to 0.06 g / L NAA, 14 g / L to 16 g / L sucrose and 6.5 g / L to 7 g / L carrageenan; the second basal medium is selected from WPM.

[0012] In one embodiment, the culture medium for seedling cultivation includes a third basal medium, and based on the third basal medium, it further contains 0.4 mg / L to 0.6 mg / L 6-BA, 14 g / L to 16 g / L sucrose, and 5 g / L to 7 g / L carrageenan; the third basal medium is selected from 1 / 2 WPM.

[0013] In one embodiment, the rooting culture medium includes a fourth basal medium, and based on the fourth basal medium, it further contains 1.8 mg / L to 2.2 mg / L IBA, 18 g / L to 22 g / L sucrose and 5 g / L to 7 g / L carrageenan; the fourth basal medium is selected from 1 / 2 WPM.

[0014] In one embodiment, the sterilization process is performed more than twice.

[0015] In one embodiment, after rooting culture, the following step is also included: transplanting the rooted seedlings obtained from the rooting culture to a seedbed for further cultivation.

[0016] In one embodiment, the substrate in the seedbed includes peat moss and vermiculite.

[0017] In one embodiment, the volume ratio of the peat moss to the vermiculite is 1:(1.8~2.2).

[0018] In one embodiment, the disinfection process includes the following steps:

[0019] The explants were rinsed with sterile water.

[0020] The explants were treated with alcohol for 4-6 seconds; and

[0021] The explants were soaked in mercuric chloride for 4 to 6 minutes.

[0022] In one embodiment, the tissue culture method satisfies at least one of the following conditions:

[0023] (1) The light intensity for the initial induction culture was 2400 lux to 3000 lux;

[0024] (2) The light intensity for proliferation culture is 2400 lux to 3000 lux; and

[0025] (3) The light intensity for rooting culture is 2400 lux to 3000 lux.

[0026] Compared with traditional technologies, this application has the following advantages:

[0027] This application investigated the effects of different basal culture media, 6-BA concentrations, and sucrose concentrations on proliferation and rooting, and improved the culture medium formula, resulting in a proliferation rate of over 2.75% and a rooting rate of over 95% for Conophytum carmichaelii tissue culture. Compared with traditional methods, the method of this application has advantages such as high proliferation rate, high rooting rate, short propagation cycle, and preservation of the superior traits of the mother plant. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 This is a schematic flowchart of a tissue culture method for *Cinnamomum camphora* according to one embodiment of this application;

[0030] Figure 2 This is a schematic diagram of the sterilization process in one embodiment of this application. Detailed Implementation

[0031] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in this application are commercially available or can be prepared by existing methods.

[0033] Some embodiments of this application provide a method for tissue culture of *Pygium wilfordii*, comprising the following steps:

[0034] Step S10: Select semi-lignified red cone scions as explants;

[0035] Step S20: Disinfect the explants;

[0036] Step S30: Primary induction culture;

[0037] Step S40: First proliferation culture;

[0038] Step S50: Sterilization treatment: including sequential seedling strengthening culture and second proliferation culture;

[0039] Step S60: Rooting culture.

[0040] In some embodiments, the culture media for the first and second proliferation cultures include a first basal culture medium, which, based on the first basal culture medium, further contains 0.4 mg / L to 0.6 mg / L 6-BA, 14 g / L to 16 g / L sucrose, and 5 g / L to 7 g / L carrageenan; the first basal culture medium is selected from WPM. It is understood that the concentrations of 6-BA, sucrose, and carrageenan in the culture media for the first and second proliferation cultures can be independent.

[0041] In some specific embodiments, the culture medium for proliferation culture includes a first basal medium, and based on the first basal medium, it further contains 0.5 mg / L 6-BA, 15 g / L sucrose and 6 g / L carrageenan, the first basal medium being selected from WPM.

[0042] It should be noted that in this application, "WPM" refers to Woody Plant Medium; "6-BA" refers to 6-Benzylaminopurine; "IBA" refers to Indole-3-Butyric Acid; and "NAA" refers to Naphthalene Acetic Acid.

[0043] In some embodiments, in step S10, semi-lignified scions of *Castanopsis fargesii* are selected as explants.

[0044] In some embodiments, in step S20, the explant is disinfected in a sterile environment.

[0045] In some embodiments, step S20, the disinfection process includes the following steps:

[0046] Rinse the explants with sterile water;

[0047] Treat explants with alcohol for 4-6 seconds; and

[0048] Soak the explants in mercuric chloride for 4 to 6 minutes.

[0049] In some specific examples, in step S20, the disinfection process includes steps S21 to S25:

[0050] Step S21: Rinse the explants 3-4 times with sterile water;

[0051] Step S22: Treat the explants with 75% pure solution for 4-6 seconds;

[0052] Step S23: Rinse the explant once with sterile water;

[0053] Step S24: Soak the explants in 0.1% mercuric chloride solution for 4 to 6 minutes;

[0054] Step S25: Rinse the explants 3-4 times with sterile water.

[0055] In some embodiments, in step S30, the culture medium for the initial induction culture includes a second basal culture medium, and based on the second basal culture medium, it further contains 1.8 mg / L to 2.2 mg / L 6-BA, 0.04 g / L to 0.06 g / L NAA, 14 g / L to 16 g / L sucrose and 6.5 g / L to 7 g / L carrageenan; the second basal culture medium is selected from WPM.

[0056] In some specific embodiments, in step S30, the primary induction culture includes a second basal culture medium, which, based on the second basal culture medium, also contains 2 mg / L 6-BA, 0.05 g / L NAA, 15 g / L sucrose and 6.8 g / L carrageenan; the second basal culture medium is selected from WPM.

[0057] In some embodiments, in step S30, the light intensity for the initial induction culture is 2400 lux to 3000 lux. Understandably, *Pinus thunbergii* stem segments can germinate under light intensities of 1000 to 3000 lux and exhibit a high survival rate. However, *Pinus thunbergii* is a strongly photophilic plant, and the induction effect under high light intensity is significantly better than under low light intensity. Therefore, this application controls the light intensity for the initial induction culture to be 2400 lux to 3000 lux.

[0058] In some specific embodiments, in step S30, the light intensity for the initial induction culture is 2500 lux.

[0059] In some embodiments, in step S40, the light intensity for proliferation culture is 2400 lux to 3000 lux.

[0060] In some specific embodiments, in step S40, the light intensity for proliferation culture is 2500 lux.

[0061] In some embodiments, in step S50, the sterilization process is performed two or more times.

[0062] In some examples, in step S50, the explants are sterilized in a sterile environment.

[0063] Specifically, in a sterile environment, the buds obtained from the propagation culture are transferred multiple times, thereby reducing endophytic bacterial contamination on the buds.

[0064] like Figure 2 As shown, a single sterilization process includes the following steps: cutting tender shoots ( Figure 2 a) Seedling cultivation ( Figure 2 (b) robust single bud ( Figure 2 c) Cut tender shoots ( Figure 2 (d) Proliferation culture ( Figure 2 (e) bud formation ( Figure 2 (f)

[0065] In some embodiments, in step S50, the culture medium for seedling cultivation includes a third basal culture medium, and based on the third basal culture medium, it further contains 0.4 mg / L to 0.6 mg / L 6-BA, 14 g / L to 16 g / L sucrose and 5 g / L to 7 g / L carrageenan; the third basal culture medium is selected from 1 / 2 WPM.

[0066] In some specific embodiments, in step S50, the culture medium for seedling cultivation includes a third basal medium, and based on the third basal medium, it also contains 0.5 mg / L 6-BA, 15 g / L sucrose and 6 g / L carrageenan; the third basal medium is selected from 1 / 2 WPM.

[0067] In some embodiments, in step S60, the rooting culture medium includes a fourth basal medium, and based on the fourth basal medium, it further contains 1.8 mg / L to 2.2 mg / L IBA, 18 g / L to 22 g / L sucrose and 5 g / L to 7 g / L carrageenan; the fourth basal medium is selected from 1 / 2 WPM.

[0068] In some specific embodiments, in step S60, the rooting culture medium includes a fourth basal medium, and based on the fourth basal medium, it also contains 2 mg / L IBA, 20 g / L sucrose and 6 g / L carrageenan; the fourth basal medium is selected from 1 / 2 WPM.

[0069] In some embodiments, in step S60, the light intensity for rooting culture is 2400 lux to 3000 lux.

[0070] In some specific embodiments, in step S60, the light intensity for rooting culture is 2500 lux.

[0071] In some embodiments, after rooting culture, step S70 is included: transplanting the rooted seedlings obtained from rooting culture to a seedbed for further cultivation.

[0072] In some implementations, the substrate in the seedbed includes peat moss and vermiculite.

[0073] In some embodiments, the volume ratio of peat moss to vermiculite is 1:(1.8~2.2).

[0074] In some specific embodiments, the volume ratio of peat moss to vermiculite is 1:2.

[0075] The method of tissue culture of Conophytum purpureus using the above method of this application has at least the following advantages: (1) The proliferation medium with the specific formula of this application can make the proliferation rate of Conophytum purpureus tissue reach more than 2.75, and the resulting plants are robust, have many buds, and have well-developed leaves; (2) The rooting medium of this application can make the rooting rate of Conophytum purpureus reach more than 95%; (3) The culture method of this application has the advantages of high propagation efficiency, short cycle, and the ability to maintain the genetic stability of the excellent traits of the mother plant.

[0076] The present application will be further described below with reference to specific embodiments and comparative examples, but these should not be construed as limiting the scope of protection of the present application. Unless otherwise specified, the raw materials involved in the following specific embodiments are all commercially available, the instruments used are all commercially available, and the processes involved are conventionally selected by those skilled in the art unless otherwise specified.

[0077] Example 1

[0078] (1) In dry, sunny autumn weather, at the red pine scion nursery (such as...) Figure 1 Select semi-lignified young shoots of *Pinus thunbergii* (as shown in Figure a) Figure 1 As shown in b), the scion was used as an explant. The scion was obliquely cut into stem segments with buds, and half of the leaves were retained for later use.

[0079] (2) Disinfection treatment

[0080] The explants were rinsed three times with sterile water in a clean bench to remove surface dust and pathogens. Then, they were treated with 75% alcohol for 5 seconds, rinsed once with sterile water, soaked in 0.1% mercuric chloride for 5 minutes, and finally rinsed three times with sterile water to reduce the amount of disinfectant residue on the stem surface.

[0081] (3) Primary induction culture

[0082] After sterilization, explants were inoculated onto WPM medium (using WPM medium as the basal medium, containing 2.0 mg / L 6-BA, 0.05 mg / L NAA, 15 g / L sucrose, and 6.8 g / L carrageenan), and then transferred to a 25°C incubator for constant temperature culture at a light intensity of 2500 lux. Newly induced axillary buds (such as...) were obtained after 30 days. Figure 1 (As shown in c).

[0083] (4) First proliferation culture

[0084] (4.1) Investigate the effects of different basal culture media on proliferation culture

[0085] 1 / 2 MS, WPM and MS were selected as basal media, and 0.3 mg / L of 6-BA was added to each basal medium. 1 cm to 2 cm long shoots were inoculated into the medium and cultured at 25 ℃ for 30 days under a light intensity of 2500 lux. Three replicates were set up for each treatment group.

[0086] The results are shown in Table 1. When 1 / 2 MS was used as the basal medium, the plant stems were weak, the leaves were yellowish, and leaf drop gradually occurred with prolonged culture time. When MS was used as the basal medium, although the plant stems were robust, vitrification occurred after multiple subcultures, and the tips of the young branches turned yellow and died. When WPM was used as the basal medium, the plants were robust, grew well, and had the highest proliferation rate, reaching 2.47. Therefore, WPM was selected as the basal medium for proliferation culture in this application.

[0087] Table 1 Effects of different basal culture media on proliferation culture

[0088]

[0089] It should be noted that the data such as proliferation rate and rooting rate in the forms of this application are all mean ± standard error; the letters "a", "b", and "c" are used to indicate the significance of differences between different treatment groups. If the two groups are labeled differently, it means that the two groups are statistically significant (P>0.05); if the two groups are labeled the same, it means that the two groups are not statistically significant (P>0.05).

[0090] (4.2) Investigate the effects of different concentrations of 6-BA on proliferation culture.

[0091] WPM was used as the basal medium, and 6-BA was added at concentrations of 0.25 mg / L, 0.5 mg / L, 1.0 mg / L and 2.0 mg / L, respectively. 1 cm to 2 cm long shoots were inoculated into the medium and cultured at a constant temperature of 25 ℃ for 30 days under a light intensity of 2500 lux. Three replicates were set up for each treatment group.

[0092] The results are shown in Table 2. Adding 6-BA promoted shoot development; with increasing concentration, the number of shoots increased while the number of leaves decreased. When the 6-BA concentration was above 1.0 mg / L, basal callus tissue increased, resulting in numerous shoots but no leaves. Furthermore, after multiple subcultures, the shoots became shorter, and the proliferation rate decreased. At a 6-BA concentration of 0.5 mg / L, the plants were robust, with numerous shoots, well-developed leaves, and a proliferation rate of 2.77. Even after multiple subcultures, the plants maintained good growth. Figure 1 d). Therefore, the concentration of 6-BA added to the basal culture medium in this application is 0.4 mg / L to 0.6 mg / L.

[0093] Table 2 Effects of different concentrations of 6-BA on proliferation culture

[0094]

[0095] (4.3) Investigate the effects of different culture media on proliferation culture.

[0096] Proliferation medium A: Modified WPM was used as the basal medium, with the addition of 3.0 mg / L 6-BA, 1.0 mg / L NAA, 15 mg / L VC, 20 mg / L aspartic acid, 30 g / L sucrose and 6.0 g / L carrageenan.

[0097] The modified WPM formula is as follows: NH4NO3 390mg / L, CaCl2·2H2O 82mg / L, MgSO4·7H2O 265mg / L, KH2PO4 170mg / L, Ca(NO3)2·4H2O 165mg / L, KI 0.83mg / L, MnSO4·H2O 22.4mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O 0.025mg / L, Na2·EDTA 33.4mg / L, FeSO4·7H2O 27.8 mg / L, inositol 200mg / L, nicotinic acid 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, thiamine hydrochloride 1.0mg / L, and glycine 2.0mg / L.

[0098] Proliferation medium B: Modified WPM was used as the basal medium, with the addition of 7.0 mg / L 6-BA, 2.5 mg / L NAA, 1.0 mg / L ZT (Zeatin), 15 mg / L VC, 20 mg / L aspartic acid, 30 g / L sucrose and 6.0 g / L carrageenan.

[0099] Proliferation medium C: WPM was used as the basal medium, with the addition of 0.5 mg / L 6-BA, 30 g / L sucrose and 6.0 g / L carrageenan.

[0100] The WPM formula is as follows: NH4NO3 400mg / L, H3BO3 6.2mg / L, CaCl2·2H2O 72.5mg / L, Ca(NO3)2·4H2O 386mg / L, MgSO4·7H2O 180mg / L, MnSO4·H2O 22.3mg / L, K2SO4 990mg, KH2PO4 170mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O 0.25mg / L, Na2·EDTA 37.3mg / L, FeSO4·7H2O 27.8mg / L. mg / L, inositol 100mg / L, nicotinic acid 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, thiamine hydrochloride 1.0mg / L, and glycine 2.0mg / L.

[0101] Young shoots of 1-2 cm in length were inoculated into the above-prepared proliferation medium A, proliferation medium B and proliferation medium C, respectively, and cultured at a constant temperature of 25℃ for 30 days under a light intensity of 2500 lux; three replicates were set up for each treatment group.

[0102] The results are shown in Table 3. The proliferation rates of *Pyracantha fortuneana* were similar across the three proliferation media, with no significant differences. However, their growth patterns differed. Specifically, plants grown in proliferation medium C were robust, with numerous clusters of buds and fully expanded leaves. In proliferation media A and B, there were more basal callus and clusters of buds, but the leaves were not fully expanded. Repeated subculturing often resulted in stunted plants, shortened internodes, and failure of leaves to emerge normally, possibly due to excessive plant hormones hindering normal plant growth. Therefore, this application uses the formulation of proliferation medium C for proliferation culture.

[0103] Table 3 Effects of different culture media on proliferation culture

[0104]

[0105] (5) Sterilization treatment

[0106] During the propagation culture process, some explant materials exhibited varying degrees of endophytic bacterial contamination. With increasing subculturing, the endophytic bacteria severely impacted plant growth, significantly reducing the propagation rate. Multiple grafting with young shoots can gradually eliminate the bacteria and re-obtain sterile seedlings. The specific steps are as follows:

[0107] According to such Figure 2 The procedure shown involves using sterile scissors to cut off the terminal 1cm length of a new shoot in a clean bench, transferring it to a seedling strengthening medium for 30 days of seedling cultivation. After the shoot has grown taller, the terminal 1cm length of the new shoot is cut off again and transferred to a proliferation medium for a second proliferation culture for 30 days to obtain clustered shoots. This process of seedling strengthening culture and second proliferation culture is repeated multiple times to obtain sterile proliferating seedlings.

[0108] The formulation of the seedling growth medium includes: 1 / 2 WPM as the basal medium, with the addition of 0.5 mg / L 6-BA, 15 g / L sucrose and 6.0 g / L carrageenan.

[0109] The culture medium formula includes: WPM + 0.5 mg / L 6-BA + 30 g / L sucrose + 6.0 g / L carrageenan.

[0110] (6) Rooting culture

[0111] (6.1) Effects of different basal culture media on rooting culture

[0112] 1 / 8 MS, 1 / 6 MS, 1 / 5 MS, 1 / 4 MS, 1 / 2 MS and 1 / 2 WPM were selected as basal media, and 1.0 mg / L IBA was added to each basal medium. Shoots about 2 cm long were inoculated into the medium and cultured at 25 ℃ for 30 days under a light intensity of 2500 lux. Three replicates were set up for each treatment group.

[0113] The results are shown in Table 4. As the concentration of inorganic salts in the basal medium decreased, the root system became stronger, the number of fibrous roots increased, and the rooting rate increased, indicating that a low-salt environment is more conducive to the formation of adventitious roots in *Pinus thunbergii*. Among them, the rooting rate of *Pinus thunbergii* was the highest when 1 / 2 WPM was used as the basal medium, reaching 74.44%, which was significantly higher than that of other treatment groups, and it is suitable as the basal medium for rooting culture.

[0114] Table 4. Effects of different basal culture media on the rooting effect of Conophytum purpureus.

[0115]

[0116] (6.2) Effects of different concentrations of IBA on rooting culture

[0117] Using 1 / 2 WPM as the basal medium, ten different mediums were prepared by adding IBA at concentrations of 0.25 mg / L, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, and 4.0 mg / L, NAA at concentrations of 0.25 mg / L, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, and 4.0 mg / L. The effects of different types and concentrations of plant hormones on rooting were investigated. Young shoots approximately 2 cm in length were inoculated into the medium and cultured at 25°C for 30 days under a light intensity of 2500 lux. Each treatment group was tested in triplicate.

[0118] The results are shown in Table 5. The overall rooting rate of *Pyracantha fortuneana* in the medium supplemented with IBA was higher than that in the medium supplemented with NAA, indicating that IBA was more effective than NAA in inducing rooting in *Pyracantha fortuneana*. Under low hormone concentrations, adventitious root germination was delayed and the root system was weak. With increasing exogenous hormone concentration, adventitious root germination was faster, and the root system became stronger and more developed, with an increased number of fibrous roots. The rooting rate was highest at 2.0 mg / L IBA, reaching 91.67%, significantly higher than other treatment groups (e.g., ...). Figure 1 (As shown in e). Therefore, this application adds IBA at a concentration of 1.8 mg / L to 2.2 mg / L to the rooting medium.

[0119] Table 5. Effects of different types and concentrations of hormones on rooting efficacy.

[0120]

[0121] (6.3) Effects of different concentrations of sucrose on rooting culture

[0122] A 1 / 2 WPM medium was used as the basal medium, supplemented with 2.0 mg / L IBA. Sucrose was added to the medium at concentrations of 0 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, and 25 g / L, respectively. Young shoots approximately 2 cm in length were inoculated into the medium and cultured at 25°C for 30 days under a light intensity of 2500 lux. Each treatment group was tested in triplicate.

[0123] The results are shown in Table 6. With increasing sucrose concentration, the rooting rate increased, the adventitious root germination cycle shortened, the number of fibrous roots increased, and the root system became more robust. The rooting rate was highest at 25 g / L, reaching 95.83%. When the sucrose concentration exceeded 25 g / L, callus formation at the base of the stem segment occurred, and the cortex became excessively swollen. Therefore, this application adds sucrose at a concentration of 18 g / L to 22 g / L to the rooting medium.

[0124] Table 6. Effects of different sucrose concentrations on rooting efficacy.

[0125]

[0126] (6.4) Effects of different culture media on rooting culture

[0127] Rooting medium A: 1 / 2 modified WPM was used as the basal medium, with 2.0 mg / L 6-BA, 1.5 mg / L NAA, 20 g / L sucrose and 6.0 g / L carrageenan added.

[0128] Rooting medium B: 1 / 2 WPM was used as the basal medium, with the addition of 2.0 mg / L 6-BA, 1.5 mg / L NAA, 20 g / L sucrose and 6.0 g / L carrageenan.

[0129] Rooting medium C: 1 / 2 WPM as the basal medium, with 2.0 mg / L IBA, 20 g / L sucrose and 6.0 g / L carrageenan added.

[0130] The results are shown in Table 7. Although the plants were robust when 6-BA and NAA were added to the rooting medium, they did not promote rooting. However, the plants grown on rooting medium C grew well, with adventitious roots germinating in about 2 weeks and a high rooting rate. Therefore, this application selected the formulation of rooting medium C for rooting culture.

[0131] Table 7 Effects of different culture media on the rooting effect of Conophytum comosum.

[0132]

[0133] (7) Domestication and transplanting

[0134] Take 30-day-old rooted seedlings from rooting bottles, loosen the caps, and place them in a sunny location to harden off for about 10 days. Remove the well-developed rooted seedlings, wash off the culture medium, and sterilize with a 500-fold dilution of carbendazim. Transplant using 32-cell trays (e.g., ...). Figure 1 As shown in Figure f), the substrate formula is peat moss:vermiculite = 1:2, with a survival rate of over 85%. In the initial transplanting stage, the seedlings should be covered with a layer of white transparent agricultural film for insulation and moisture retention. A 75%–80% shade net should then be placed outside the film. New roots will develop in the seedlings after 1–2 weeks. When the seedlings are 3–4 weeks old and growing vigorously, water and ventilate appropriately, remove the insulation film, and apply 0.1% diluted fertilizer weekly. When the seedlings reach 5cm in height, transplant them into 8cm*10cm black plastic bags using yellow clay as the substrate, and replace the shade net with one providing 40%–60% shading. Later, when the seedlings reach 15cm–20cm in height, they can grow under natural environmental conditions.

[0135] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0136] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A method for tissue culture of *Conophytum rubrum*, characterized in that, Includes the following steps: Semi-lignified *Conophytum purpureus* scions were selected as explants. The scions were obliquely cut into stem segments with buds, retaining half a leaf; and The explants were subjected to disinfection, primary induction culture, first proliferation culture, sterilization treatment, and rooting culture in sequence. The sterilization treatment includes sequential seedling cultivation and a second proliferation culture; The culture media for both the first and second proliferation cultures consisted of the following components: a first basal medium, with 0.5 mg / L 6-BA, 30 g / L sucrose, and 6 g / L carrageenan added as a base; the first basal medium was WPM, which consisted of the following components: NH4NO3 400 mg / L, H3BO3 6.2 mg / L, CaCl2·2H2O 72.5 mg / L, Ca(NO3)2·4H2O 386 mg / L, MgSO4·7H2O 180 mg / L, MnSO4·H2O 22.3 mg / L, K2SO4 990 mg, KH2PO4 170 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.25 mg / L, and Na2·EDTA. 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, inositol 100 mg / L, nicotinic acid 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, thiamine hydrochloride 1.0 mg / L, and glycine 2.0 mg / L; The culture medium for the initial induction culture consisted of the following components: a second basal medium, with 1.8 mg / L~2.2 mg / L 6-BA, 0.04 g / L~0.06 g / L NAA, 14 g / L~16 g / L sucrose and 6.5 g / L~7 g / L carrageenan added based on the second basal medium; the second basal medium was WPM; The culture medium for cultivating robust seedlings consists of the following components: a third basal medium, with 0.4 mg / L to 0.6 mg / L 6-BA, 14 g / L to 16 g / L sucrose, and 5 g / L to 7 g / L carrageenan added based on the third basal medium; the third basal medium is 1 / 2 WPM. The rooting culture medium consists of the following components: a fourth basal medium, with 1.8 mg / L to 2.2 mg / L IBA, 18 g / L to 22 g / L sucrose and 5 g / L to 7 g / L carrageenan added based on the fourth basal medium; the fourth basal medium is 1 / 2 WPM.

2. The tissue culture method for *Cinnamomum camphora* according to claim 1, characterized in that, The sterilization process is performed more than twice.

3. The method for tissue culture of *Conophytum rubrum* according to any one of claims 1 to 2, characterized in that, After rooting culture, the following steps are also included: transplanting the rooted seedlings obtained from rooting culture to a seedbed for further cultivation.

4. The tissue culture method for *Cinnamomum camphora* according to claim 3, characterized in that, The substrate in the seedbed includes peat moss and vermiculite.

5. The tissue culture method for *Cinnamomum camphora* according to claim 4, characterized in that, The volume ratio of the peat soil to vermiculite is 1:(1.8~2.2).

6. The method for tissue culture of *Conophytum rubrum* according to any one of claims 1-2 and 4-5, characterized in that, Disinfection treatment includes the following steps: The explants were rinsed with sterile water. The explants were treated with alcohol for 4-6 seconds; and The explants were soaked in mercuric chloride for 4 to 6 minutes.

7. The method for tissue culture of *Conophytum rubrum* according to any one of claims 1-2 and 4-5, characterized in that, The tissue culture method satisfies at least one of the following conditions: (1) The light intensity for the initial induction culture was 2400 lux to 3000 lux; (2) The light intensity for proliferation culture is 2400 lux to 3000 lux; and (3) The light intensity for rooting culture is 2400 lux to 3000 lux.

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