Tissue culture propagation method of cinnamomum japonicum var. japonicum no. 1

Abstract

This invention relates to the field of plant tissue culture technology, specifically to a method for tissue culture propagation of *Cinnamomum camphora* species *Jilong 1*. The method includes the following steps: pre-treating young leaves of the *Jilong 1* mother plant as explants; inoculating the pre-treated explants into an induction medium for ultrasound-assisted induction culture to obtain callus tissue; inoculating the callus tissue into a differentiation medium for differentiation culture to obtain adventitious buds; inoculating the adventitious buds into a proliferation medium for proliferation induction culture to obtain clustered buds; and removing robust single buds from the clustered buds of *Jilong 1* camphor tree and inoculating them into a rooting medium for induced rooting culture to obtain *Jilong 1* seedlings. The provided seedling propagation method does not damage the rare mother tree, has a high callus induction rate and a high propagation success rate, and can achieve large-scale propagation of *Jilong 1* seedlings.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a tissue culture propagation method for the plant *Cinnamomum camphora* 'Jilong No. 1'. Background Technology

[0002] Jilong No. 1 is a new plant variety of the genus *Cinnamomum* Tree, recognized and authorized by the National Forestry and Grassland Administration on May 13, 2022, and has been issued a new variety rights certificate. First discovered in 2012 by researchers at the Ji'an Forestry Research Institute, its leaf shape, leaf tip, and leaf veins are unique and novel. It is the first leaf in the genus *Cinnamomum* of the Lauraceae family to be nearly round, without a leaf tip, and with the midrib not extending to the leaf margin. The entire leaf is curved and spoon-shaped, making it highly ornamental. Leaf essential oil from Jilong No. 1 and its offspring was tested by the Jiangxi Provincial Testing Center and found to contain high levels of precious Chinese medicinal materials and high-grade spice natural borneol. Therefore, Jilong No. 1 is a superior new *Cinnamomum* plant variety with both ornamental and economic value, and there is huge market demand.

[0003] Currently, the propagation of Jilong No. 1 is mainly done through cuttings. However, cutting propagation is highly seasonal and requires a large number of cuttings from the mother plant, which causes great damage to the rare mother trees. The mother plants of Jilong No. 1 are rare and precious, the number of cuttings is small, and the success rate of cuttings is low. Tissue culture propagation method has the advantages of not damaging rare mother trees, not being restricted by location or season, and being able to produce all year round.

[0004] It is imperative to achieve the propagation of Jilong 1 through tissue culture propagation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a tissue culture method for *Cinnamomum camphora* species *Jilong No. 1*. The provided method does not damage the rare mother tree, has a high callus induction rate and a high propagation success rate, and can achieve large-scale propagation of *Cinnamomum camphora* seedlings.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a tissue culture propagation method for the genus *Cinnamomum*, namely, *Jilong No. 1*, comprising the following steps: S1. Young leaves of the mother plant of Camphor genus Jilong No. 1 were used as explants for pretreatment. S2. The pretreated explants were inoculated into an induction medium for ultrasound-assisted induction culture to obtain callus tissue. The induction medium consisted of modified MS medium No. 1 with the following added ingredients: 25-35 g / L sucrose, 4-6 g / L gelatin, 1-3 g / L chitosan, 0.5-2 g / L nano-activated carbon, 2-4 mg / L 6-benzylaminopurine (6-BA), 0.1-0.3 mg / L naphthaleneacetic acid (NAA), and 0.1-0.3 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D). S3. Inoculate the callus tissue into differentiation medium and carry out differentiation culture to obtain adventitious shoots; the differentiation medium is modified MS medium No. 1 with the addition of 25-35 g / L sucrose, 4-6 g / L gelatin, 1-3 g / L chitosan, 0.5-2 g / L nano-activated carbon, 0.8-2.0 mg / L 6-benzylaminopurine, and 0.1-0.3 mg / L naphthaleneacetic acid; S4. Inoculate the adventitious buds into the proliferation medium and induce proliferation culture to obtain clustered buds; the proliferation medium is modified MS medium No. 1 with the addition of 25-35 g / L sucrose, 4-6 g / L gelatin, 1-3 g / L chitosan, 0.5-2 g / L nano-activated carbon, 0.5-1.5 mg / L 6-benzylaminopurine, and 0.1-0.3 mg / L indoleacetic acid (IAA). S5. Take robust single buds from the clustered buds of Camphor Tree No. 1 and inoculate them into rooting medium to induce rooting culture and obtain seedlings of Camphor Tree No. 1. The rooting medium is modified MS medium No. 2 with the addition of 25-35 g / L sucrose, 4-6 g / L gelatin, 1-3 g / L chitosan, 0.5-2 g / L nano activated carbon, 0.1-0.3 mg / L indoleacetic acid, and 0.1-0.3 mg / L indolebutyric acid (IBA).

[0007] This application utilizes young leaves of the new Cinnamomum camphora cultivar Jilong 1 as explants, and sequentially performs pretreatment, callus induction, adventitious bud induction, bud proliferation culture, and root induction culture to obtain seedlings of Jilong 1. Callus induction is the most critical and time-consuming process. Through long-term observation of the growth habits and micronutrient requirements of the new Cinnamomum camphora cultivar Jilong 1, and considering the complex chemical composition, high oil content, and tendency to browning of the selected explants, this invention modifies the MS medium to obtain a callus induction medium. Gelatin is used instead of carrageenan, and chitosan and nano-activated carbon are added to improve the callus induction rate and reduce browning. Kalanamycin sulfate and vancomycin hydrochloride are added for antibacterial purposes, and the concentrations of auxins and cytokinins are screened according to requirements. This method successfully obtains callus tissue from the new Cinnamomum camphora cultivar Jilong 1. This method directly uses young leaves of Jilong 1 as explants without damaging the rare mother tree.

[0008] Further, the pretreatment method described in S1 involves taking young leaves from the mother plant of the *Cinnamomum camphora* species *Jilong No. 1* as explants, cutting them into small pieces, sealing them, and then treating them in the dark at 3-7℃ for 10-12 hours. Afterward, the leaves are removed, cleaned with sterile water, disinfected sequentially with alcohol and mercuric chloride, rinsed with sterile water, and then soaked in a pretreatment solution for 30-60 minutes. The pretreatment solution includes 1-10 μmol / L melatonin and 5-15 μmol / L strigolactone. Addressing the characteristics of *Jilong No. 1* mother plant young leaf explants being prone to browning, exhibiting slow callus growth, and having a low effective callus rate, this method pretreats the explants with specific concentrations of melatonin and strigolactone before inoculation. At specific concentrations, the two have a synergistic effect; after pretreatment, the callus induction rate and effective callus rate are significantly increased, and the browning index is reduced.

[0009] Furthermore, the ultrasound-assisted induction culture method involves inoculating pretreated explants into an induction medium and culturing them in the dark at 26-30℃ for 14-16 days, followed by low-light culture at 24-26℃ and a light intensity of 1000-1500 Lux. For the first three days of dark culture, an additional 1 minute of ultrasound treatment at 20-40 kHz and 50 W is applied daily. By first performing ultrasound-assisted dark culture followed by two stages of low-light culture, under suitable conditions, the induction time can be shortened with minimal impact on the callus induction success rate.

[0010] Furthermore, the induction medium described in S2 also includes 40 µg / ml kanamycin sulfate and 40 µg / ml vancomycin hydrochloride.

[0011] Furthermore, the differentiation medium described in S3 also includes 100 µg / ml cefotaxime sodium.

[0012] Furthermore, the specific method for differentiation culture in S3 is to culture it under the conditions of a culture temperature of 26-28℃ and a light intensity of 1400-1600 Lux.

[0013] Furthermore, the specific method for proliferation induction culture described in S4 is to culture the culture at a temperature of 26-28℃ and a light intensity of 2000-3000 Lux.

[0014] Furthermore, the specific method for inducing rooting culture described in S5 is to culture the plants under conditions of a culture temperature of 24-26℃ and a light intensity of 2000-3000 Lux.

[0015] Further, the formulation of the No. 1 modified MS medium is as follows: ammonium nitrate 825 mg / L, potassium nitrate 2850 mg / L, calcium chloride 440 mg / L, magnesium sulfate heptahydrate 555 mg / L, potassium dihydrogen phosphate 255 mg / L, sodium dihydrogen phosphate 85 mg, boric acid 9.3 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, potassium iodide 0.83 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, disodium EDTA 37.3 mg / L, inositol 150 mg / L, nicotinic acid 1.0 mg / L, vitamin B6 1.0 mg / L, vitamin B1 0.2 mg / L, and glycine 4.0 mg / L.

[0016] Furthermore, the formulation of the No. 2 modified MS medium is as follows: potassium nitrate 950 mg / L, ammonium nitrate 825 mg / L, calcium chloride dihydrate 220 mg / L, magnesium sulfate heptahydrate 185 mg / L, potassium dihydrogen phosphate 85 mg / L, sodium dihydrogen phosphate 85 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, potassium iodide 0.83 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, disodium EDTA 37.3 mg / L, inositol 100 mg / L, nicotinic acid 0.5 mg / L, vitamin B6 0.5 mg / L, vitamin B1 0.1 mg / L, and glycine 2.0 mg / L.

[0017] Compared with the prior art, the beneficial effects of the present invention are: 1. This application utilizes young leaves of the mother plant of the new Cinnamomum camphora cultivar Jilong 1 as explants, and sequentially performs pretreatment, callus induction, adventitious bud induction, bud proliferation culture, and root induction culture to obtain seedlings of Jilong 1. A modified MS medium was used to obtain a callus induction medium. This method successfully obtains callus tissue from the new Cinnamomum camphora cultivar Jilong 1. Using young leaves of Jilong 1 as explants, this method causes no damage to the rare mother tree. By pretreating the explants with specific concentrations of melatonin and strigolactone before inoculation, followed by ultrasonic-assisted dark culture followed by two stages of low-light culture, the callus induction rate and effective callus rate are significantly improved, the browning index is low, and the induction time is shortened.

[0018] 2. By modifying the culture medium for different stages of adventitious bud induction, bud proliferation, and rooting, and by selectively adding gelatin, chitosan, and nano-activated carbon, and by screening for the required concentrations of auxin and cytokinin, the method of this invention promotes subsequent differentiation, proliferation, and rooting of callus tissue. This method effectively improves the propagation efficiency and economic benefits of Camphor Tree No. 1 and can be applied to the mass production of Camphor Tree No. 1 seedlings, promoting the development and utilization of Camphor Tree No. 1 resources. Attached Figure Description

[0019] Figure 1 A flowchart of the tissue culture propagation method of the camphor tree species Jilong No. 1 according to the present invention. Detailed Implementation

[0020] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] This application utilizes young leaves from the mother plant of the new Cinnamomum camphora cultivar Jilong 1 as explants, and sequentially performs pretreatment, callus induction, adventitious bud induction, bud proliferation culture, and root induction culture to obtain seedlings of Cinnamomum camphora cultivar Jilong 1. The culture process is as follows: Figure 1 The image shown illustrates different stages of tissue culture propagation of *Cinnamomum camphora* seedlings, specifically including the following steps: [Image showing different stages of tissue culture propagation of *Cinnamomum camphora* seedlings, including the following steps...] S1. Young leaves of the mother plant of Camphor genus Jilong No. 1 were used as explants for pretreatment. S2. The pretreated explants were inoculated into an induction medium and subjected to ultrasound-assisted induction culture to obtain callus tissue. The induction medium consisted of modified MS medium No. 1 with the following added ingredients: 25-35 g / L sucrose, 4-6 g / L gelatin, 1-3 g / L chitosan, 0.5-2 g / L nano-activated carbon, 2-4 mg / L 6-benzylaminopurine, 0.1-0.3 mg / L naphthaleneacetic acid, and 0.1-0.3 mg / L 2,4-dichlorophenoxyacetic acid. S3. Inoculate the callus tissue into differentiation medium and carry out differentiation culture to obtain adventitious shoots; the differentiation medium is modified MS medium No. 1 with the addition of 25-35 g / L sucrose, 4-6 g / L gelatin, 1-3 g / L chitosan, 0.5-2 g / L nano-activated carbon, 0.8-2.0 mg / L 6-benzylaminopurine, and 0.1-0.3 mg / L naphthaleneacetic acid; S4. Adventitious shoots are inoculated into proliferation medium and induced to grow into clustered shoots. The proliferation medium is modified MS medium No. 1 with the following added ingredients: 25-35 g / L sucrose, 4-6 g / L gelatin, 1-3 g / L chitosan, 0.5-2 g / L nano-activated carbon, 0.5-1.5 mg / L 6-benzylaminopurine, and 0.1-0.3 mg / L indoleacetic acid. S5. Take robust single buds from the clustered buds of Camphor Tree No. 1 and inoculate them into rooting medium to induce rooting culture and obtain seedlings of Camphor Tree No. 1. The rooting medium is modified MS medium No. 2 with the addition of 25-35 g / L sucrose, 4-6 g / L gelatin, 1-3 g / L chitosan, 0.5-2 g / L nano activated carbon, 0.1-0.3 mg / L indoleacetic acid, and 0.1-0.3 mg / L indolebutyric acid.

[0022] The compositions of Modified MS medium No. 1 and Modified MS medium No. 2 used in the following embodiments and comparative examples of the present invention are as follows: Modified MS medium No. 1: Ammonium nitrate 825 mg / L, potassium nitrate 2850 mg / L, calcium chloride 440 mg / L, magnesium sulfate heptahydrate 555 mg / L, potassium dihydrogen phosphate 255 mg / L, sodium dihydrogen phosphate 85 mg, boric acid 9.3 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, potassium iodide 0.83 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, disodium EDTA 37.3 mg / L, inositol 150 mg / L, nicotinic acid 1.0 mg / L, vitamin B6 1.0 mg / L, vitamin B1 0.2 mg / L, glycine 4.0 mg / L.

[0023] Modified MS medium No. 2: Potassium nitrate 950 mg / L, ammonium nitrate 825 mg / L, calcium chloride dihydrate 220 mg / L, magnesium sulfate heptahydrate 185 mg / L, potassium dihydrogen phosphate 85 mg / L, sodium dihydrogen phosphate 85 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, potassium iodide 0.83 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, disodium EDTA 37.3 mg / L, inositol 100 mg / L, nicotinic acid 0.5 mg / L, vitamin B6 0.5 mg / L, vitamin B1 0.1 mg / L, glycine 2.0 mg / L.

[0024] Unless otherwise specified, all methods described herein are conventional methods, and all raw materials described herein are available from publicly available commercial sources.

[0025] The method of this application will be further described below with reference to specific embodiments: Example 1: Callus Induction Young leaves of the new Cinnamomum camphora cultivar Jilong No. 1 were used as explants. They were cut into 1.0cm x 1.0cm square pieces with a brand-new scalpel blade, placed in a sealed container, and refrigerated at 5°C for 12 hours. Afterward, the explants were cleaned with sterile water, soaked in 70% alcohol for 0.5 minutes, rinsed twice with sterile water, disinfected with 0.1% mercuric chloride for 5 minutes, and rinsed four times with sterile water. The treated explants of Jilong No. 1 were then dried with filter paper, and two horizontal and two vertical incisions were made on the underside of the leaf, without cutting through the leaf.

[0026] To compare the callus induction results of treatment groups 1-6 (P1-P6) on different induction media, leaf abaxial contact medium was inoculated into containers containing different induction media. Ten containers were inoculated for each treatment group, with three leaflets inoculated per container. The containers were covered with opaque material. The culture conditions for each treatment group were as follows: P1: The induction medium consisted of MS medium supplemented with 30 g / L sucrose, 7 g / L carrageenan, 40 µg / ml kanamycin sulfate, 40 µg / ml vancomycin hydrochloride, 2 mg / L 6-BA, 0.1 mg / L NAA, and 0.1 mg / L 2,4-D, and incubated in the dark at 28°C.

[0027] P2: The induction medium consisted of 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L activated carbon nanotubes, 40 µg / ml kanamycin sulfate, 40 µg / ml vancomycin hydrochloride, 2 mg / L 6-BA, 0.1 mg / L NAA, and 0.1 mg / L 2,4-D added to modified MS medium No. 1, and cultured in the dark at 28°C.

[0028] P3: The induction medium consisted of MS medium supplemented with 30 g / L sucrose, 7 g / L carrageenan, 40 µg / ml kanamycin sulfate, 40 µg / ml vancomycin hydrochloride, 3 mg / L 6-BA, 0.2 mg / L NAA, and 0.2 mg / L 2,4-D, and incubated in the dark at 28°C.

[0029] P4: The induction medium consisted of 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L activated carbon nanotubes, 40 µg / ml kanamycin sulfate, 40 µg / ml vancomycin hydrochloride, 3 mg / L 6-BA, 0.2 mg / L NAA, and 0.2 mg / L 2,4-D added to modified MS medium No. 1, and cultured in the dark at 28°C.

[0030] P5: The induction medium consisted of MS medium supplemented with 30 g / L sucrose, 7 g / L carrageenan, 40 µg / ml kanamycin sulfate, 40 µg / ml vancomycin hydrochloride, 4 mg / L 6-BA, 0.3 mg / L NAA, and 0.3 mg / L 2,4-D, and incubated in the dark at 28°C.

[0031] P6: The induction medium consisted of 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L activated carbon nanotubes, 40 µg / ml kanamycin sulfate, 40 µg / ml vancomycin hydrochloride, 4 mg / L 6-BA, 0.3 mg / L NAA, and 0.3 mg / L 2,4-D added to modified MS medium No. 1, and cultured in the dark at 28°C.

[0032] The callus ratio, effective callus ratio, and browning index of each treatment group were statistically analyzed and calculated. The callus ratio = number of calluses formed / number of samples × 100%, and the effective callus ratio = number of effective calluses / number of samples × 100%. The browning index was evaluated by having multiple evaluators observe the samples and record the browning level of each sample according to the grading criteria in Table 1. The browning index was calculated as follows: browning index = [Σ(browning score of each level × number of samples at that level) / (maximum browning score × total number of samples)] × 100%. The effects of different culture media and hormone ratios on callus induction are shown in Table 2. Table 1: Browning Grading Scoring

[0033] Table 2: Effects of different culture media and hormone ratios on callus induction

[0034] Table 2 shows that, using young leaves of the mother plant of the new Cinnamomum camphora variety Jilong 1 as explants, all treatment groups were able to induce callus tissue for Jilong 1. Under the same hormone ratio, the optimized culture medium showed a higher callus induction rate and effective callus rate, and a lower browning index compared to the unoptimized medium, while the overall induction culture time was similar.

[0035] The optimal hormone ratio was 6-BA 3.0 + NAA 0.2 + 2,4-D 0.2. While increasing the hormone dosage resulted in a higher callus induction rate, it also reduced the effective callus ratio, leading to loose, water-soaked, and vitrified callus. In treatment group 4 (P4), the optimized culture medium resulted in an effective callus ratio of 33.3% and a browning index of 52.3%, indicating a still low induction rate and a high browning index. The following section will use the culture conditions from treatment group 4 (P4) to further improve the callus induction conditions and increase the induction rate while reducing the browning index.

[0036] Example 2: Callus Induction Young leaves of the new Cinnamomum camphora cultivar Jilong 1 were used as explants. They were cut into 1.0cm x 1.0cm square pieces with a brand-new scalpel and placed in a sealed container in a refrigerator at 5°C for 12 hours. After removal, the explants were cleaned with sterile water, soaked in 70% alcohol for 0.5 minutes, rinsed twice with sterile water, disinfected with 0.1% mercuric chloride for 5 minutes, and rinsed four times with sterile water. Different pretreatment solutions were then applied for 45 minutes each. After drying the treated explants with filter paper, two horizontal and two vertical cuts were made on the underside of the leaves, without cutting through the leaf. The underside of the leaves was inoculated into induction medium (the induction medium for treatment group 4 (P4) in Example 1). Ten bottles were inoculated for each treatment group, with three leaves inoculated into each bottle. The inoculation container was wrapped with opaque material and incubated in the dark at 28°C.

[0037] The effects of different pretreatment solutions on callus induction were compared among treatment groups 1-6 (Y1-Y6). The results are shown in Table 3. The composition of the pretreatment solutions for each treatment group is as follows: Y1: The pretreatment solution consists of 5 μmol / L melatonin; Y2: The pretreatment solution consists of 5 μmol / L strigolactone; Y3: The pretreatment solution consists of 10 μmol / L strigolactone; Y4: The pretreatment solution consists of 5 μmol / L melatonin and 5 μmol / L strigolactone; Y5: The pretreatment solution consists of 5 μmol / L melatonin and 10 μmol / L strigolactone; Y6: The pretreatment solution consists of 5 μmol / L melatonin and 15 μmol / L strigolactone; Table 3: Effects of different pretreatment solutions on callus induction

[0038] Table 3 shows that when young leaves of the new Cinnamomum camphora cultivar Jilong 1 were used as explants, pretreatment with a solution before inoculation induced callus formation in all treatment groups. Treatment group 1 (Y1) used melatonin alone as the pretreatment solution, resulting in improved callus induction and effective callus rates compared to treatment group 4 (P4) in Example 1, while reducing the browning index. Treatment groups 2-3 (Y2-Y3) used strigolactone alone as the pretreatment solution, showing no significant changes in callus induction and effective callus rates or browning index compared to treatment group 4 (P4) in Example 1. In treatment groups 4-6 (Y4-Y6), melatonin 5 + strigolactone 5-15 was used as a pretreatment solution, and the callus induction rate and effective callus rate were significantly improved compared with treatment group 1 (Y1). In treatment group 5 (Y5), melatonin 5 + strigolactone 10 was used as a pretreatment solution with the best effect, and the callus induction rate and effective callus rate could reach 90.0%, while the browning index was reduced to 34.2%, but the overall induction time was still relatively long.

[0039] Example 3: Callus Induction Young leaves of the new Cinnamomum camphora cultivar Jilong 1 were used as explants. They were cut into 1.0cm x 1.0cm square pieces using a brand-new scalpel, placed in a sealed container, and refrigerated at 5°C for 12 hours. Using a brand-new scalpel and refrigeration prevented polyphenol oxidase from oxidizing the cut surfaces, reducing the risk of browning. The explants were then cleaned with sterile water, soaked in 70% alcohol for 0.5 minutes, rinsed twice with sterile water, disinfected with 0.1% mercuric chloride for 5 minutes, and rinsed four times with sterile water. They were then pretreated with a solution of 5μmol / L melatonin and 10μmol / L strigolactone for 45 minutes. After absorbing excess water with filter paper, two horizontal and two vertical incisions were made on the underside of the leaves, but without cutting through the leaf. The leaf back contact medium was inoculated into the induction medium (the induction medium of treatment group 4 (P4) in Example 1). Ten bottles were inoculated for each treatment group, and three leaves were inoculated into each bottle. The inoculation container bottle was wrapped with opaque material.

[0040] Table 4 shows a comparison of the effects of different culture methods on callus induction in each treatment group. The culture methods for each treatment group are as follows: C1: After 15 days of dark culture at 28℃, remove the opaque material covering the inoculation container and transfer it to a low-light culture at 25℃ with a light intensity of 1200 Lux. C2: After 15 days of dark culture at 28℃, remove the opaque material covering the inoculation container and transfer it to a low-light culture at 25℃ and a light intensity of 1200 Lux. For the first 3 days of dark culture, apply an additional 20kHz, 50W ultrasonic treatment for 1 minute each day.

[0041] C3: After 15 days of dark culture at 28℃, remove the opaque material covering the inoculation container and transfer it to a low-light culture at 25℃ and a light intensity of 1200 Lux. For the first 3 days of dark culture, apply an additional 1 min of ultrasonic treatment at 40 kHz and 50 W per day.

[0042] C4: After 15 days of dark culture at 28℃, remove the opaque material covering the inoculation container and transfer it to a low-light culture at 25℃ and a light intensity of 1200 Lux. For the first 3 days of dark culture, apply an additional 1 min of ultrasonic treatment at 60 kHz and 50 W per day.

[0043] C5: After 15 days of dark culture at 28℃, remove the opaque material covering the inoculation container and transfer it to a low-light culture at 25℃ and a light intensity of 1200 Lux. For the first 3 days of dark culture, apply an additional 20kHz, 50W sonication for 5 minutes each day.

[0044] C6: After 15 days of dark culture at 28℃, remove the opaque material covering the inoculation container and transfer it to a low-light culture at 25℃ and a light intensity of 1200 Lux. For the first 3 days of dark culture, apply an additional 40kHz, 50W ultrasonic treatment for 5 minutes each day.

[0045] Table 4: Effects of different culture conditions on callus induction

[0046] Table 4 shows that, using young leaves from the mother plant of the new Cinnamomum genus 'Jilong 1' as explants, treatment group 1 (C1) underwent 15 days of dark culture at 28℃, followed by low-light culture at 25℃ and 1200 Lux, resulting in a shorter induction time but a higher browning index. Treatment groups 2-3 (C2-C3) underwent 15 days of dark culture at 28℃, followed by culture at 25℃ and 1200 Lux, and simultaneously underwent 1 min of ultrasound treatment at 20-40 kHz for the first three days of dark culture. This shortened the induction time while achieving a callus induction rate and an effective callus rate of 93.3%, with the browning index controlled at 34.2-36.4%. Treatment group 4 (C4), with an ultrasound frequency of 60 kHz, showed significantly lower callus induction and effective callus rates, and a significantly higher browning index, indicating that the high ultrasound power in the early stages may have caused tissue damage to the explants. In treatment groups 5-6 (C5-C6), the ultrasound time was 5 minutes. The callus induction rate and effective callus rate were both low, and the browning index was increased, indicating that excessive ultrasound time may also cause explant tissue damage.

[0047] Example 4: Induction of adventitious buds Effective callus tissue was divided into small clumps approximately 0.3cm × 0.3cm square and inoculated into differentiation medium. Ten bottles were inoculated for each treatment group, with one clump inoculated per bottle. The callus induction results of treatment groups 1-10 (numbered B1-B10) on different differentiation media were compared. The following differentiation media were used, and the cultures were incubated at 27℃ and 1500 Lux light intensity. The effects of different media and hormone ratios on adventitious shoot differentiation are shown in Table 5. The composition of the differentiation media for each treatment group is as follows: B1: The differentiation medium was MS medium supplemented with 30 g / L sucrose, 7 g / L carrageenan, 100 µg / ml cefotaxime sodium, 0.8 mg / L 6-BA, and 0.1 mg / L NAA. B2: The differentiation medium was prepared by adding 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano-activated carbon, 100 µg / ml cefotaxime sodium, 0.8 mg / L 6-BA, and 0.1 mg / L NAA to modified MS medium No. 1. B3: The differentiation medium was MS medium supplemented with 30 g / L sucrose, 7 g / L carrageenan, 100 µg / ml cefotaxime sodium, 1.4 mg / L 6-BA, and 0.1 mg / L NAA. B4: The differentiation medium was prepared by adding 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano-activated carbon, 100 µg / ml cefotaxime sodium, 1.4 mg / L 6-BA, and 0.1 mg / L NAA to modified MS medium No. 1. B5: The differentiation medium was MS medium supplemented with 30 g / L sucrose, 7 g / L carrageenan, 100 µg / ml cefotaxime sodium, 2 mg / L 6-BA, and 0.1 mg / L NAA. B6: The differentiation medium was prepared by adding 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano-activated carbon, 100 µg / ml cefotaxime sodium, 2 mg / L 6-BA, and 0.1 mg / L NAA to modified MS medium No. 1. B7: The differentiation medium was MS medium supplemented with 30 g / L sucrose, 7 g / L carrageenan, 100 µg / ml cefotaxime sodium, 1.4 mg / L 6-BA, and 0.3 mg / L NAA. B8: The differentiation medium was prepared by adding 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano-activated carbon, 100 µg / ml cefotaxime sodium, 1.4 mg / L 6-BA, and 0.3 mg / L NAA to modified MS medium No. 1. B9: The differentiation medium was MS medium supplemented with 30 g / L sucrose, 7 g / L carrageenan, 100 µg / ml cefotaxime sodium, 2 mg / L 6-BA, and 0.3 mg / L NAA. B10: The differentiation medium was prepared by adding 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano-activated carbon, 100 µg / ml cefotaxime sodium, 2 mg / L 6-BA, and 0.3 mg / L NAA to modified MS medium No. 1. Table 5: Effects of different culture media and hormone ratios on adventitious shoot differentiation

[0048] As shown in Table 5, under the same hormone ratio, the total number of effective buds and the overall bud condition of the optimized basal culture medium used in this application are better than those before optimization. It should be noted that the concentration of cytokinin and auxin needs to be well controlled during the differentiation culture of adventitious buds. Too high a concentration can easily lead to a decline in the quality of callus and adventitious buds. Treatment group 4 (B4) is the best.

[0049] Example 5: Bud proliferation Adventitious bud clusters with a small amount of callus were divided into groups of 3 buds each and inoculated into proliferation medium. Ten bottles were inoculated for each treatment group, with one cluster inoculated per bottle. The results of adventitious bud proliferation in treatment groups 1-6 (numbered Z1-Z6) on different proliferation media were compared. The following proliferation media were used, and the cultures were grown at 27℃ and 2500 Lux. The effects of different hormone ratios on adventitious bud proliferation are shown in Table 6. The composition of the proliferation media for each treatment group is as follows: Z1: The proliferation medium consisted of modified MS medium No. 1 with the addition of 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano-activated carbon, 0.5 mg / L 6-BA, and 0.1 mg / L IAA. Z2: The proliferation medium was prepared by adding 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano-activated carbon, 1.0 mg / L 6-BA, and 0.1 mg / L IAA to modified MS medium No. 1. Z3: The proliferation medium was prepared by adding 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano-activated carbon, 1.5 mg / L 6-BA, and 0.1 mg / L IAA to modified MS medium No. 1. Z4: The proliferation medium was prepared by adding 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano-activated carbon, 0.5 mg / L 6-BA, and 0.3 mg / L IAA to modified MS medium No. 1. Z5: The proliferation medium was prepared by adding 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano-activated carbon, 1.0 mg / L 6-BA, and 0.3 mg / L IAA to modified MS medium No. 1. Z6: The proliferation medium consisted of modified MS medium No. 1 with the addition of 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano-activated carbon, 1.5 mg / L 6-BA, and 0.3 mg / L IAA. Table 6: Effects of different hormone ratios on adventitious bud proliferation

[0050] As shown in Table 6, the method of this application can achieve the proliferation of adventitious shoots with a high proliferation rate when the hormone ratio is appropriate. It should be noted that the concentration of cytokinin and auxin needs to be well controlled during the proliferation culture process. Too high a concentration can easily lead to a decrease in callus and proliferation quality. The method of treatment group 2 (Z2) is the best.

[0051] Example 6: Root Induction Robust single shoots with a height greater than 2.5 cm were selected from the shoot clusters and inoculated into rooting medium. Ten bottles were inoculated for each treatment group, with two shoots inoculated per bottle. The root induction results of treatment groups 1-6 (numbered G1-G6) on different rooting media were compared. The following rooting media were used and cultured at a temperature of 25℃ and a light intensity of 2500 Lux. The effects of different media and hormone ratios on root induction are shown in Table 7. The composition of the rooting media for each treatment group is as follows: G1: The rooting medium is 1 / 2 MS medium supplemented with 30 g / L sucrose, 7 g / L carrageenan, 0.1 mg / L IAA, and 0.1 mg / L IBA; G2: The rooting medium is modified MS medium No. 2 with the addition of 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano activated carbon, 0.1 mg / L IAA, and 0.1 mg / L IBA. G3: The rooting medium is 1 / 2 MS medium supplemented with 30 g / L sucrose, 7 g / L carrageenan, 0.2 mg / L IAA, and 0.2 mg / L IBA; G4: The rooting medium is modified MS medium No. 2 with the addition of 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano activated carbon, 0.2 mg / L IAA, and 0.2 mg / L IBA. G5: The rooting medium is 1 / 2 MS medium supplemented with 30 g / L sucrose, 7 g / L carrageenan, 0.3 mg / L IAA, and 0.3 mg / L IBA; G6: The rooting medium is modified MS medium No. 2 with the addition of 30 g / L sucrose, 5 g / L gelatin, 2 g / L chitosan, 1 g / L nano activated carbon, 0.3 mg / L IAA, and 0.3 mg / L IBA. Table 7: Effects of different culture media and hormone ratios on root induction

[0052] As shown in Table 7, under the same hormone ratio, the rooting ratio and rooting condition of the optimized proliferation medium used in this application are better than those of the conventional 1 / 2MS medium. During the rooting culture process, the concentration of auxin needs to be well controlled. Too high a concentration can easily lead to a small amount of callus at the junction of the seedling stems. The results of treatment group 4 (G4) are the best.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and not to limit them; although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of this application or equivalent substitutions can be made to some technical features, all of which should be covered within the scope of the technical solutions claimed in this application.

Claims

1. A tissue culture propagation method for *Cinnamomum camphora* species *Jilong No. 1*, characterized in that, Includes the following steps: S1. Young leaves of the mother plant of Camphor genus Jilong No. 1 were used as explants for pretreatment. S2. The pretreated explants were inoculated into an induction medium and subjected to ultrasound-assisted induction culture to obtain callus tissue. The induction medium consisted of modified MS medium No. 1 with the following added ingredients: 25-35 g / L sucrose, 4-6 g / L gelatin, 1-3 g / L chitosan, 0.5-2 g / L nano-activated carbon, 2-4 mg / L 6-benzylaminopurine, 0.1-0.3 mg / L naphthaleneacetic acid, and 0.1-0.3 mg / L 2,4-dichlorophenoxyacetic acid. S3. Inoculate the callus tissue into differentiation medium and carry out differentiation culture to obtain adventitious shoots; the differentiation medium is modified MS medium No. 1 with the addition of 25-35 g / L sucrose, 4-6 g / L gelatin, 1-3 g / L chitosan, 0.5-2 g / L nano-activated carbon, 0.8-2.0 mg / L 6-benzylaminopurine, and 0.1-0.3 mg / L naphthaleneacetic acid; S4. Adventitious shoots are inoculated into proliferation medium and induced to grow into clustered shoots. The proliferation medium is modified MS medium No. 1 with the following added ingredients: 25-35 g / L sucrose, 4-6 g / L gelatin, 1-3 g / L chitosan, 0.5-2 g / L nano-activated carbon, 0.5-1.5 mg / L 6-benzylaminopurine, and 0.1-0.3 mg / L indoleacetic acid. S5. Take robust single buds from the clustered buds of Camphor Tree No. 1 and inoculate them into rooting medium to induce rooting culture and obtain seedlings of Camphor Tree No.

1. The rooting medium is modified MS medium No. 2 with the addition of 25-35 g / L sucrose, 4-6 g / L gelatin, 1-3 g / L chitosan, 0.5-2 g / L nano activated carbon, 0.1-0.3 mg / L indoleacetic acid, and 0.1-0.3 mg / L indolebutyric acid.

2. The tissue culture propagation method for *Cinnamomum camphora* species *Jilong No. 1* according to claim 1, characterized in that, The pretreatment method described in S1 is as follows: young leaves of the mother plant of the genus Cinnamomum camphora, Jilong No. 1, are taken as explants, cut into small pieces, sealed, and treated in the dark at 3-7℃ for 10-12 hours. After being taken out, they are cleaned with sterile water, disinfected with alcohol and mercuric chloride in sequence, rinsed with sterile water, and then soaked in a pretreatment solution for 30-60 minutes. The pretreatment solution includes 1-10 μmol / L melatonin and 5-15 μmol / L strigolactone.

3. The tissue culture propagation method for *Cinnamomum camphora* species *Jilong No. 1* according to claim 1, characterized in that... The method of ultrasound-assisted induction culture is as follows: the pretreated explants are inoculated into the induction culture medium and cultured in the dark at 26-30℃ for 14-16 days, and then transferred to low light culture at 24-26℃ and light intensity of 1000-1500 Lux; on the first to third days of dark culture, an additional 20-40kHz, 50W ultrasound treatment is applied for 1 min each day.

4. The tissue culture propagation method for *Cinnamomum camphora* species *Jilong No. 1* according to claim 1, characterized in that, The No. 1 induction medium described in S2 also includes 40 µg / ml kanamycin sulfate and 40 µg / ml vancomycin hydrochloride.

5. The tissue culture propagation method for *Cinnamomum camphora* species *Jilong No. 1* according to claim 1, characterized in that, The differentiation medium described in S3 also includes 100 µg / ml cefotaxime sodium.

6. The tissue culture propagation method for *Cinnamomum camphora* species *Jilong No. 1* according to claim 1, characterized in that, The specific method for differentiation culture in S3 is to culture it at a temperature of 26-28℃ and a light intensity of 1400-1600 Lux.

7. The tissue culture propagation method for *Cinnamomum camphora* plant *Jilong No. 1* according to claim 1, characterized in that, The specific method for proliferation induction culture described in S4 is to culture the culture at a temperature of 26-28℃ and a light intensity of 2000-3000 Lux.

8. The tissue culture propagation method for *Cinnamomum camphora* species *Jilong No. 1* according to claim 1, characterized in that, The specific method for inducing rooting culture described in S5 is to culture the plants at a temperature of 24-26℃ and a light intensity of 2000-3000 Lux.

9. The tissue culture propagation method for *Cinnamomum camphora* species *Jilong No. 1* according to claim 1, characterized in that, The formulation of the No. 1 modified MS medium is as follows: ammonium nitrate 825 mg / L, potassium nitrate 2850 mg / L, calcium chloride 440 mg / L, magnesium sulfate heptahydrate 555 mg / L, potassium dihydrogen phosphate 255 mg / L, sodium dihydrogen phosphate 85 mg, boric acid 9.3 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, potassium iodide 0.83 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, disodium EDTA 37.3 mg / L, inositol 150 mg / L, nicotinic acid 1.0 mg / L, vitamin B6 1.0 mg / L, vitamin B1 0.2 mg / L, and glycine 4.0 mg / L.

10. The tissue culture propagation method for *Cinnamomum camphora* species *Jilong No. 1* according to claim 1, characterized in that, The formulation of the No. 2 modified MS medium is as follows: potassium nitrate 950 mg / L, ammonium nitrate 825 mg / L, calcium chloride dihydrate 220 mg / L, magnesium sulfate heptahydrate 185 mg / L, potassium dihydrogen phosphate 85 mg / L, sodium dihydrogen phosphate 85 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, potassium iodide 0.83 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, ferrous sulfate heptahydrate 27.8 mg / L, disodium EDTA 37.3 mg / L, inositol 100 mg / L, nicotinic acid 0.5 mg / L, vitamin B6 0.5 mg / L, vitamin B1 0.1 mg / L, and glycine 2.0 mg / L.

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