Method for inducing adventitious roots of cinnamomum camphora

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to an induction method of adventitious roots of cinnamomum camphora. According to the method disclosed by the invention, the adventitious root induction rate of the adventitious root of the cinnamomum camphora is remarkably improved by establishing a sterile system of the cinnamomum camphora, culturing cluster buds and sterile seedlings, promoting rooting of tissue culture seedlings, inhibiting browning, enabling the adventitious buds to extend and inducing the adventitious root through three ways, so that the problems of shortage of borneol resources and high market demand are solved; the method for inducing the adventitious roots of the cinnamomum camphora can be used for industrial production, and sufficient raw materials are provided for extraction of borneol; according to the method, large-scale culture of the adventitious roots of the cinnamomum camphora is achieved, a large number of pharmaceutical raw materials can be obtained at low cost in a short time, and an integrated production system can be formed with the pharmaceutical industry. The technology has important practical significance for promoting the development of the cinnamomum camphora industry, expanding the application of the pharmaceutical field, constructing a rare and endangered traditional Chinese medicinal material breeding technology system and realizing sustainable development and utilization.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing adventitious roots of Cinnamomum camphora var. borneol. BACKGROUND

[0002] Cinnamomum camphora var. borneol is an intraspecific variant of Cinnamomum camphora, which was first discovered in Ji'an, Jiangxi Province in the 1980s. Cinnamomum camphora var. borneol is not only a rare medicinal material, but also a high-grade spice and an important chemical raw material, and is known as plant gold. It is currently used as the best plant resource for extracting dextro-rotatory borneol due to its highest content of essential oil and dextro-rotatory borneol in the essential oil.

[0003] Borneol (natural camphor) has extremely high medicinal, industrial and economic values. As the core component of traditional emergency drugs (such as An Gong Niuhuang Pill), it can open the orifices, awaken the mind, clear heat and relieve pain, and has antibacterial and anti-inflammatory effects, and can significantly enhance the transdermal absorption of external use drugs. Industrially, its unique aroma ranks among the “four great famous fragrances” and is widely used in high-end spices and daily chemical products. However, natural borneol resources are extremely scarce: Cinnamomum camphora var. borneol in Southeast Asia is on the verge of exhaustion due to overexploitation, and the annual yield of Cinnamomum camphora var. borneol in China is far lower than the market demand (more than 170 kinds of preparations containing borneol are included in the 2020 edition of Chinese Pharmacopoeia). The imbalance between supply and demand leads to the price of natural borneol being more than 10 times that of synthetic products, and synthetic borneol is limited in use due to the presence of toxic isoborneol (ophthalmic drugs and other products require natural raw materials). At present, China is alleviating the resource crisis through asexual reproduction technology of Cinnamomum camphora var. borneol (such as large-scale planting in Jiangxi Province), but breakthroughs in yield bottlenecks are urgently needed.

[0004] Adventitious roots refer to roots produced by plants on non-root organs (especially on damaged or flooded plants) or callus. It can promote plant growth and development or regenerate complete plants from damaged plants, and is widely used in cutting and tissue culture of forestry and horticulture. Compared with callus, adventitious roots are easier to collect, have high differentiation degree, strong regenerative ability, good stability, and strong ability to synthesize secondary metabolites; compared with hairy roots, it does not need Agrobacterium rhizogenes infection for formation, is non-transgenic, and has high safety. The culture of adventitious roots is simple and easy to operate, and is easy to scale up for the production of plant secondary metabolites.

[0005] In recent years, research on the use of adventitious root culture to produce secondary metabolites has been increasingly active, and has been successfully applied in medicinal plants such as Panax ginseng and Panax notoginseng. However, the culture of adventitious roots of Cinnamomum camphora var. borneol has not been reported. Therefore, it is of great importance to establish and optimize the induction system of adventitious roots to realize large-scale industrial production. Through tissue culture technology, the occurrence of adventitious roots of Cinnamomum camphora var. borneol is induced, and the rapid batch production of active ingredients of Cinnamomum camphora var. borneol is realized, which can solve the problem of market demand at home and abroad. Therefore, it is urgent to develop a method for inducing adventitious roots of Cinnamomum camphora var. borneol to solve the above technical problems. Summary of the Invention

[0006] To address the above problems, the present invention aims to provide a method for inducing adventitious roots of camphor tree.

[0007] The technical content of this invention is as follows: This invention provides a method for inducing adventitious roots of camphor tree, comprising the following steps: 1) Establishment of a camphor camphor sterile system New shoots emerging in the current year were selected as explants. They were disinfected by soaking in 0.2% sodium hypochlorite solution for 10-15 minutes, then in 0.1% mercuric chloride solution for 5-10 minutes, and then rinsed thoroughly with sterile water. The contamination rate after disinfection was 13%-24%. 2) Preparation of aseptic clustered buds of camphor tree The explants obtained in step 1) were inoculated into the bud-inducing medium to obtain camphor buds; The budding induction medium comprises: MS solid medium supplemented with 0.5-2.0 mg / L 6-BA and 0.05-0.5 mg / L NAA; 3) Preparation of sterile camphor tree seedlings The obtained camphor tree buds were inoculated into a sterile seedling culture medium to obtain camphor tree sterile seedlings; The sterile seedling culture medium comprises: MS solid medium supplemented with 0.5~2.0 mg / L 6-BA, 0.05~0.5 mg / L NAA, 0.1~0.5 mg / L GA3 and 0.05%~0.2% (w / v) activated carbon; 4) Induced adventitious roots Approach 1: Induction of adventitious roots via callus tissue pathway a) Take the sterile seedling stem segment obtained in step 3) and inoculate it into the callus induction medium for culture to induce callus production; The callus induction medium comprises: B5 solid medium supplemented with 0.5-2 mg / L 2,4-D, 0.1-1 mg / L 6-BA, 0.1-0.3 mg / L TDZ, 100-500 mg / L Pro and 1-2 g / L PVP; b) The induced callus tissue was inoculated into an adventitious root induction medium and cultured to induce the generation of adventitious roots; The adventitious root induction medium comprises: 1 / 2MS solid medium supplemented with 2-6 mg / L IBA, 0.1-0.5 mg / L NAA, 0.1-0.3 mg / L KT and 1-2 g / L PVP.

[0008] Approach 2: Direct induction of adventitious roots from leaves The sterile seedling leaves obtained in step 3) are cut, several incisions are made on the leaves, and the leaves are inoculated into the adventitious root direct induction medium to induce adventitious roots; The adventitious root direct induction medium comprises: 1 / 2MS solid medium, 0.5-2.5 mg / L IBA, 0.1-0.5 mg / L NAA, 0.1-0.3 mg / L KT and 1-2 g / L PVP.

[0009] Route three: stem segment directly inducing adventitious roots The sterile seedling stems (length 1.0±0.2 cm) obtained in step 3) are inoculated into the adventitious root direct induction medium to induce adventitious roots. The adventitious root direct induction medium comprises: 1 / 2MS solid medium, 0.5-2.5 mg / L IBA, 0.1-0.5 mg / L NAA, 0.1-0.3 mg / L KT and 1-2 g / L PVP.

[0010] The beneficial effects of the present application are as follows: The method for inducing the adventitious roots of Cinnamomum camphora in the present application promotes rooting of the tissue culture seedlings, inhibits browning, and makes the adventitious buds elongate, and then induces the adventitious roots through three routes, thereby significantly improving the rooting induction rate of the adventitious roots of Cinnamomum camphora, solving the problem of shortage of camphor resources and large market demand, inducing uniform, rapid and feasible cultures, and providing sufficient raw materials for extraction of camphor. The present application realizes large-scale cultivation of the adventitious roots of Cinnamomum camphora, and a large amount of pharmaceutical raw materials can be obtained at low cost in a short period of time, and an integrated production system can be formed with the pharmaceutical industry. The present application has important practical significance for promoting the development of the Cinnamomum camphora industry, expanding the application in the pharmaceutical field, and constructing a breeding technology system for rare and endangered Chinese medicinal materials and realizing sustainable development and utilization. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Sterile clustered buds of Cinnamomum camphora; Figure 2 Sterile seedlings of Cinnamomum camphora; Figure 3 Cinnamomum camphora callus; Figure 4 Adventitious roots induced by Cinnamomum camphora callus; Figure 5 Adventitious roots induced by Cinnamomum camphora leaves; Figure 6 Adventitious roots induced by Cinnamomum camphora stem segments. DETAILED DESCRIPTION

[0012] The application will be further described in detail below with specific examples and drawings. It should be understood that these examples are only used to illustrate the application and not to limit the protection scope of the application. After reading the application, those skilled in the art can make various equivalent modifications of the application, which fall within the scope defined by the appended claims.

[0013] Unless otherwise specified, all raw materials and reagents of the application are conventional market raw materials and reagents.

[0014] Abbreviations involved: IBA: indole-3-butyric acid; 6-BA: 6-benzylaminopurine; NAA: naphthalene acetic acid; KT: kinetin; 2,4-D: 2,4-dichlorophenoxyacetic acid; PVP: polyvinylpyrrolidone; Pro: proline.

[0015] Example 1 A method for inducing uncertain roots of Drynaria fortunei 1) Establishment of a sterile system of Drynaria fortunei Select the current year's growth of healthy and disease-free Drynaria fortunei tender shoots with a length of about 8 cm to ensure that the explants have good growth potential and inducibility. Cut the Drynaria fortunei tender shoots into 1-2 cm small sections as explants. In the clean bench, immerse them in a container containing 0.2% sodium hypochlorite for 12 minutes, then take them out and immerse them in 0.1% mercury chloride (HgCl2) solution for 6 minutes. During the disinfection process, constantly shake the explants gently to ensure that the disinfectant uniformly covers the surface of the explants. Finally, rinse them with sterile water for 5 times, each time for no less than 30 seconds to ensure that the residual disinfectant is removed to prevent the disinfectant from affecting the subsequent culture process. Dry the surface moisture of the disinfected explants with sterile filter paper, then inoculate them into the basic MS solid medium without any hormone and observe them for 5-7 days. The culture conditions are temperature 25℃±2℃, light intensity 1500-2500 lx, and light time 16 hours / day. The contamination rate is 15%, which is the acceptable threshold. A sterile system is successfully established.

[0016] 2) Preparation of sterile multiple shoots of Drynaria fortunei Sterilized explants were inoculated into MS solid medium containing 1.5 mg / L 6-BA and 0.05 mg / L NAA, i.e. shoot induction medium, using forceps. Four explants were inoculated into each bottle. The culture bottles were placed in a culture room at a temperature of 25±2℃, a light intensity of 2000~3000 lx, and a light duration of 16 h / d for 50 days. The average number of shoots was 25.5. Depend on Figure 1 It is evident that this culture medium has a significant effect on inducing clustered buds on camphor tree stem segments. The resulting clustered buds not only have a high proliferation rate, a large number of effective buds, and a fast growth rate, but also show no obvious vitrification phenomenon.

[0017] 3) Preparation of sterile camphor tree seedlings When the bud clusters grew to 2-3 cm, they were cut from the induction medium and transferred to MS solid medium (sterile medium) supplemented with 1.5 mg / L 6-BA, 0.3 mg / L NAA, 0.2 mg / L GA3, and 0.1% (w / v) activated carbon. Four buds were inoculated per bottle. After culturing under the same conditions for another 6 weeks, the resulting sterile seedlings showed vigorous growth, dark green leaves, and well-developed root systems.

[0018] Depend on Figure 2 It is evident that this culture medium significantly enhanced the subculture proliferation efficiency of clustered shoots and the quality of sterile seedlings. The sterile seedlings obtained after subculture (culture conditions: 25±1℃, 12 h light / 12 h dark, light intensity 2000 lx) exhibited robust growth, reaching a height of 5-8 cm, with thick stems (approximately 2-3 mm in diameter), short and uniform internodes, and no excessive elongation or weakness. The leaves were a deep green, characterized by their spread-out shape, thick texture, and uniform dark green color, without yellowing or chlorosis spots. The root system was well-developed, robust, and highly vigorous, with each plant capable of sprouting 3-5 roots 4-6 cm long, demonstrating strong nutrient absorption and plant fixation capabilities, laying a solid foundation for survival and growth after subsequent substrate transplanting.

[0019] Studies have shown that adding activated carbon to the culture medium not only promotes rooting of tissue culture seedlings but also inhibits browning by adsorbing harmful substances. Meanwhile, reducing the concentration of 6-BA and adding a low concentration of NAA is beneficial for adventitious shoot elongation.

[0020] Example 2 A method for inducing adventitious roots of camphor tree The bud induction medium for step 2) is MS solid medium containing 2.0 mg / L 6-BA and 0.5 mg / L NAA; The sterile medium of step 3) is MS solid medium added with 2.0 mg / L 6-BA, 0.5 mg / L NAA, 0.5 mg / L GA3 and 0.2% (w / v) activated carbon; The other steps are the same as those of Example 1.

[0021] Example 3 A method for inducing adventitious roots of Cinnamomum camphora The sterile medium of step 3) is MS solid medium added with 2.0 mg / L 6-BA, 0.5 mg / L NAA, 0.5 mg / L GA3 and 0.2% (w / v) activated carbon; The sterile medium of step 3) is MS solid medium added with 2.0 mg / L 6-BA, 0.5 mg / L NAA, 0.5 mg / L GA3 and 0.2% (w / v) activated carbon; The other steps are the same as those of Example 1.

[0022] Comparative Example 1 Comparative Example 1 differs from Example 1 in that the sterile medium of step 2) is MS solid medium without hormones.

[0023] The other steps are the same as those of Example 1.

[0024] Comparative Example 2 Comparative Example 2 differs from Example 1 in that the sterile medium of step 3) is MS solid medium added with 1.5 mg / L 6-BA, 0.3 mg / L NAA and 0.2 mg / L GA3.

[0025] The other steps are the same as those of Example 1.

[0026] Table 1 Comparison of effects of different treatments on induction of Cinnamomum camphora shoots and growth of aseptic seedlings

[0027] From Table 1, it can be seen that the best effect of the induction stage of the cluster buds is MS solid medium containing 1.5 mg / L 6-BA and 0.05 mg / L NAA, the induction rate is 93.2%, and the bud proliferation number is 25.5, which is significantly higher than that of Example 2 (2.0 mg / L 6-BA+0.5 mg / L NAA, the proliferation number is 17.4), Example 3 (0.5 mg / L 6-BA+0.3 mg / L NAA, the proliferation number is 3.2) and Comparative Example 1 (no hormone, the induction rate is 23.6%, and the proliferation number is 2.1), which shows that the ratio of 6-BA and NAA presents a dose-dependent regulation characteristic on the induction efficiency of the cluster buds, and has a significant influence on the induction rate and the proliferation number of the cluster buds, and the appropriate combination of 6-BA and NAA is the key to high induction rate and high proliferation of the cluster buds, and too low or too high concentration will inhibit the growth.

[0028] The sterile seedling culture stage: the MS solid medium added with 1.5 mg / L 6-BA, 0.3 mg / L NAA, 0.2 mg / L GA3 and 0.1% activated carbon shows the best performance, the sterile seedling increment is 5.5±0.4 cm, the average stem diameter is 2.8 mm, and the average root number is 5.8, the growth is healthy (plant height 5-8 cm, stem diameter 2-3 mm), the leaf is dark green, and the root system is developed (3-5 per plant, 4-6 cm long), which is significantly better than that of Example 2 (4.1 cm, 2.6 mm, 5.6) and Example 3 (2.5 cm, 2.4 mm, 4.8); although the cluster bud induction rate of Comparative Example 2 (without activated carbon) is high (92%), the seedling increment is only 1.7 cm, the stem diameter is 1.5 mm, and the root number is 0, which clearly shows that GA3 (0.2 mg / L) has a promoting effect on the bud elongation, and activated carbon (0.1%) has a decisive influence on the rooting (can adsorb harmful substances and inhibit browning, which is a necessary factor for the induction of adventitious roots).

[0029] In summary: the formula of Example 1 (cluster bud induction: 1.5 mg / L 6-BA+0.05 mg / L NAA; sterile seedling culture: 1.5 mg / L 6-BA+0.3 mg / L NAA+0.2 mg / L GA3+0.1% activated carbon) has the best comprehensive effect, in which the low concentration of 6-BA (1.5 mg / L) promotes the proliferation of the cluster buds, the NAA concentration is suitable for the stage requirement (low concentration in the induction period and slightly high concentration in the culture period), the low concentration of GA3 avoids the elongation, and the activated carbon is indispensable, which can provide technical support for the large-scale tissue culture and rapid propagation of Cinnamomum camphora.

[0030] Example 4 Induction of adventitious roots by callus approach a) Take healthy stem segments from sterile camphor tree seedlings obtained in Example 1 and aseptically cut them into 1 cm segments (the cut should be smooth to reduce tissue damage). Place the stem segments flat in B5 solid medium containing 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 0.2 mg / L TDZ, 300 mg / L Pro, and 1.0 g / L PVP, i.e., callus induction medium. Place 5 segments per bottle, gently press the base to ensure contact with the medium, and incubate in the dark at 25°C for 2 weeks. The cut ends of the stem segments will swell to form callus (induction rate 93.8%). Depend on Figure 3 As can be seen, the cut surface of the stem segment swells and gradually forms callus tissue; after subculture, the callus tissue grows rapidly and increases in volume significantly, with an overall pale yellow or milky white color. At this stage, the callus tissue not only increases in volume significantly, but also exhibits vigorous cell division and maintains good differentiation potential, providing a high-quality material basis for subsequent experimental steps such as organ differentiation (e.g., shoot induction, root induction) or cell suspension culture.

[0031] The callus induction culture medium includes, but is not limited to: B5 solid culture medium supplemented with 0.5-2 mg / L 2,4-D, 0.1-1 mg / L 6-BA, 0.1-0.3 mg / L TDZ, 100-500 mg / L Pro and 1-2 g / L PVP; b) Cut the induced callus into 5×5 mm pieces and transfer them to the surface of 1 / 2 MS medium containing 2.0 mg / L IBA, 0.1 mg / L NAA, 0.1 mg / L KT and 1.0 g / L PVP (5 pieces evenly distributed per bottle), i.e. adventitious root induction medium, and continue to incubate in the dark at 25°C for 5 weeks.

[0032] Depend on Figure 4 It is evident that adventitious roots successfully differentiated on the surface of the callus tissue during the cultivation process. These adventitious roots are milky white, uniform in shape, and grow vigorously, fully demonstrating that the callus tissue has a strong root differentiation ability.

[0033] Example 5 The difference between Example 5 and Example 4 is that the adventitious root induction medium for callus tissue is 1 / 2 MS medium containing 2.0 mg / L IBA, 0.3 mg / L NAA, 0.2 mg / L KT and 1.0 g / L PVP.

[0034] Example 6 The difference between Example 6 and Example 4 is that the adventitious root induction medium for callus tissue is 1 / 2 MS medium containing 2.0 mg / L IBA, 0.5 mg / L NAA, 0.3 mg / L KT and 1.0 g / L PVP.

[0035] Example 7 Example 7 differs from Example 4 in that the adventitious root induction medium for the callus is 1 / 2MS medium with 4.0 mg / L IBA, 0.1 mg / L NAA, 0.2 mg / L KT, and 1.0 g / L PVP.

[0036] Example 8 Example 8 differs from Example 4 in that the adventitious root induction medium for the callus is 1 / 2MS medium with 4.0 mg / L IBA, 0.3 mg / L NAA, 0.3 mg / L KT, and 1.0 g / L PVP.

[0037] Example 9 Example 9 differs from Example 4 in that the adventitious root induction medium for the callus is 1 / 2MS medium with 4.0 mg / L IBA, 0.5 mg / L NAA, 0.1 mg / L KT, and 1.0 g / L PVP.

[0038] Example 10 Example 10 differs from Example 4 in that the adventitious root induction medium for the callus is 1 / 2MS medium with 6.0 mg / L IBA, 0.1 mg / L NAA, 0.3 mg / L KT, and 1.0 g / L PVP.

[0039] Example 11 Example 11 differs from Example 4 in that the adventitious root induction medium for the callus is 1 / 2MS medium with 6.0 mg / L IBA, 0.3 mg / L NAA, 0.1 mg / L KT, and 1.0 g / L PVP.

[0040] Example 12 Example 12 differs from Example 4 in that the adventitious root induction medium for the callus is 1 / 2MS medium with 6.0 mg / L IBA, 0.5 mg / L NAA, 0.2 mg / L KT, and 1.0 g / L PVP.

[0041] Comparative Example 3 Comparative Example 3 differs from Example 4 in that the callus induction medium of step a) is MS solid medium with 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 0.2 mg / L TDZ, 300 mg / L Pro, and 1.0 g / L PVP.

[0042] Comparative Example 4 Comparative Example 4 differs from Example 4 in that the callus induction medium of step a) is 1 / 2MS solid medium containing 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 0.2 mg / L TDZ, 300 mg / L Pro and 1.0 g / L PVP.

[0043] Comparative Example 5 Comparative Example 5 differs from Example 4 in that the callus induction medium of step a) is WPM solid medium containing 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 0.2 mg / L TDZ, 300 mg / L Pro and 1.0 g / L PVP.

[0044] Table 2 Comparison of callus induction rate, browning rate and growth state of experimental examples and comparative examples

[0045] As shown in Table 2, the callus of Experimental Example 4 shows the best performance, with the highest induction rate (93.8%) and the lowest browning rate (9.8%), and the callus is milky white and grows fast. In the comparative examples, the groups with high browning rate (such as 30.2% in Comparative Example 3 and 27.6% in Comparative Example 5) have low induction rate (both < 86%) and slow growth. The browning rate of Comparative Example 4 is slightly high (16.1%), the induction rate is slightly low (91.0%) but close to that of Experimental Example 4, and the growth speed is moderate. In summary, the treatment condition of Experimental Example 4 is the best, and the browning rate is negatively correlated with the callus induction effect.

[0046] Comparative Example 6 Comparative Example 6 differs from Example 4 in that the adventitious root induction medium of step b) is 1 / 2MS medium containing 2.0 mg / L IBA, 0.1 mg / L NAA and 0.1 mg / L KT, i.e. the adventitious root induction medium.

[0047] Comparative Example 7 Comparative Example 7 differs from Example 4 in that the adventitious root induction medium of step b) is 1 / 2MS medium containing 2.0 mg / L IBA and 0.1 mg / L NAA, i.e. the adventitious root induction medium.

[0048] Table 3 Rooting rate of callus pathway induced adventitious roots

[0049] As can be seen from Table 3, in terms of rooting rate, Example 8 (adventitious root induction medium of 4.0 mg / L IBA+0.3 mg / L NAA+0.3 mg / L KT+1.0 g / L PVP+1 / 2MS) performs best, with a rooting rate of 92.5%, followed by Example 9 (4.0 mg / L IBA+0.5 mg / L NAA+0.1 mg / L KT+1.0 g / L PVP+1 / 2MS, 89.8%), and Example 12 (61.1%) with the highest concentration of IBA (6.0 mg / L) has the lowest rooting rate; in terms of root morphology, the average number of roots (4.3, 3.9 per block), root length (2.0-4.2 cm, 2.0-3.8 cm), and root thickness (1.3, 1.2 mm) of Examples 8 and 9 are significantly better than those of other groups, while the root thickness (0.5 mm) and root length (0.5-2.0 cm) of Examples 11 and 12 (6.0 mg / L IBA) are the worst; in terms of browning rate, all examples containing 1.0 g / L PVP have a relatively low browning rate (12%-19%), with Example 8 (12%) being the lowest, while Comparative Example 6 (without PVP) has a browning rate as high as 45%, fully demonstrating the key role of PVP in inhibiting browning of callus.

[0050] Further analysis of the effects of hormones found that the appropriate concentration of IBA is 4.0 mg / L, and too high (6.0 mg / L) will inhibit the formation of root primordia; NAA needs to be coordinated with IBA, and 0.3-0.5 mg / L can significantly increase the average number of roots; KT optimizes root morphology when it is 0.1-0.3 mg / L, both promoting the increase of root number (such as 0.3 mg / L KT in Example 8) and improving root thickness (such as 0.1 mg / L KT in Example 9).

[0051] In summary, the optimal formula for inducing adventitious roots from callus of Cinnamomum loureiroi is 1 / 2MS+4.0 mg / L IBA+0.3 mg / L NAA+0.3 mg / L KT+1.0 g / L PVP, which has a high rooting rate (92.5%), a robust root morphology (an average of 4.3 per block, root length of 2.0-4.2 cm, and root thickness of 1.3 mm), and a low browning rate (12%), providing reliable technical support for the induction of adventitious roots from callus of Cinnamomum loureiroi and large-scale propagation.

[0052] Example 13 Leaf Direct Induction of Adventitious Roots Select the healthy leaves of Example 1 growing vigorously, and carefully draw 3-5 longitudinal wounds on the leaf surface with a sterilized scalpel. Subsequently, the treated leaves are transferred to the surface of 1 / 2MS solid medium containing 2.0 mg / L IBA, 0.3 mg / L NAA, 0.2 mg / L KT and 1.0 g / L PVP (5 leaves per bottle), i.e. the direct induction medium of adventitious roots, and gently press the leaves (especially the wound) to make them fully contact with the medium for nutrient absorption. The treated leaves are placed in a constant temperature incubator at 25°C for dark culture. After 21 days of culture, multiple adventitious roots are induced at the wound of the leaves; the average number of roots per leaf is 14.2, and the rooting rate is 92.3%.

[0053] Example 14 Direct induction of adventitious roots from leaves Select the healthy leaves of Example 1 growing vigorously, and carefully draw 3-5 longitudinal wounds on the leaf surface with a sterilized scalpel. Subsequently, the treated leaves are transferred to the surface of 1 / 2MS solid medium containing 2.0 mg / L IBA, 0.3 mg / L NAA, 0.2 mg / L KT and 1.0 g / L PVP (5 leaves per bottle), i.e. the direct induction medium of adventitious roots, and gently press the leaves (especially the wound) to make them fully contact with the medium for nutrient absorption. The treated leaves are placed in a constant temperature incubator at 25°C for dark culture. After 21 days of culture, multiple adventitious roots are induced at the wound of the leaves; the average number of roots per leaf is 14.2, and the rooting rate is 92.3%.

[0054] From Figure 5 It can be seen that multiple adventitious roots are induced at the wound of the leaves; the average number of roots per leaf is 14.2, the rooting rate is 92.3%, the root color is pure and milky white, the morphology is full and the tip is transparent, the vigor is vigorous, and the roots can smoothly elongate.

[0055] Example 15 Direct induction of adventitious roots from leaves Select the healthy leaves of Example 1 growing vigorously, and carefully draw 3-5 longitudinal wounds on the leaf surface with a sterilized scalpel. Subsequently, the treated leaves are transferred to the surface of 1 / 2MS solid medium containing 2.0 mg / L IBA, 0.3 mg / L NAA, 0.2 mg / L KT and 1.0 g / L PVP (5 leaves per bottle), i.e. the direct induction medium of adventitious roots, and gently press the leaves (especially the wound) to make them fully contact with the medium for nutrient absorption. The treated leaves are placed in a constant temperature incubator at 25°C for dark culture. After 21 days of culture, multiple adventitious roots are induced at the wound of the leaves; the average number of roots per leaf is 14.2, and the rooting rate is 92.3%.

[0056] Comparative Example 8 The difference between Comparative Example 8 and Example 13 is that the adopted adventitious root direct induction medium does not contain PVP, and the others are the same.

[0057] The average number of roots per leaf is 6.3, and the rooting rate is 62.4%.

[0058] Example 16 Stem segment direct induction of adventitious roots The healthy stem segments of the aseptic Cinnamomum camphora seedlings in Example 1 are selected, and are cut into 1.0±0.2 cm small segments under aseptic conditions (the cut surface is smooth). The stem segments are placed horizontally in 1 / 2MS solid medium containing 2.0 mg / L IBA, 0.3 mg / L NAA, 0.2 mg / L KT and 1.0 g / L PVP, i.e. the adventitious root direct induction medium, 5 segments are inoculated in each bottle, and the base is lightly pressed to ensure contact with the medium. After 21 days of culture at 25°C in the dark, the cut surface of the stem segment induces the generation of adventitious roots. The average number of roots per stem segment is 5.2, and the rooting rate is 85.8%.

[0059] From Figure 6 It can be seen that a plurality of milky white adventitious roots are differentiated from the cut surface of the stem segment, the root system is strong, the diameter is about 0.2-0.3 cm, and the length can reach 3-6 cm. The average number of roots per stem segment is 5.2, and the rooting rate is 85.8%.

[0060] Example 17 Stem segment direct induction of adventitious roots The healthy stem segments of the aseptic Cinnamomum camphora seedlings in Example 1 are selected, and are cut into 1.0±0.2 cm small segments under aseptic conditions (the cut surface needs to be kept smooth to reduce tissue damage). The stem segments are placed horizontally in 1 / 2MS solid medium containing 1.0 mg / L IBA, 0.2 mg / L NAA, 0.2 mg / L KT and 1.0 g / L PVP, i.e. the adventitious root direct induction medium, 5 segments are inoculated in each bottle, and the base is lightly pressed to ensure contact with the medium. After 21 days of culture at 25°C in the dark, the cut surface of the stem segment induces the generation of adventitious roots. The average number of roots per stem segment is 4.4, and the rooting rate is 71.5%.

[0061] Example 18 Stem segment direct induction of adventitious roots The healthy stem segments of the aseptic Cinnamomum camphora seedlings in Example 1 are selected, and are cut into 1.0±0.2 cm small segments under aseptic conditions (the cut surface is smooth). The stem segments are placed horizontally in 1 / 2MS solid medium containing 0.5 mg / L IBA, 0.1 mg / L NAA, 0.1 mg / L KT and 1.0 g / L PVP, i.e. the adventitious root direct induction medium, 5 segments are inoculated in each bottle, and the base is lightly pressed to ensure contact with the medium. After 21 days of culture at 25°C in the dark, the cut surface of the stem segment induces the generation of adventitious roots. The average number of roots per stem segment is 4.8, and the rooting rate is 65.6%.

[0062] Comparative Example 9 Comparative Example 9 differs from Example 17 in that the direct induction medium employed did not contain PVP, otherwise unchanged.

[0063] An average of 3.2 roots per stem segment were produced, with a rooting rate of 57.1%.

Claims

1. A method for inducing camphor tree adventitious roots, characterized by, The method comprises the following steps: 1) Establishing a sterile system of Cinnamomum camphora 2) Preparing sterile shoots of Cinnamomum camphora The explants obtained in step 1) are inoculated in a shoot induction medium to obtain shoots of Cinnamomum camphora; The shoot induction medium comprises 6-BA and NAA; 3) Preparing sterile seedlings of Cinnamomum camphora The shoots obtained are inoculated in a sterile seedling medium to obtain sterile seedlings of Cinnamomum camphora; The sterile seedling medium comprises 6-BA, NAA, GA3 and activated carbon; 4) Inducing adventitious roots Pathway one: inducing adventitious roots through a callus a) The sterile seedlings obtained in step 3) are inoculated in a callus induction medium to induce callus; The callus induction medium comprises 2,4-D, 6-BA, TDZ, Pro and PVP; b) The induced callus is inoculated in an adventitious root induction medium to induce adventitious roots; The adventitious root induction medium comprises IBA, NAA, KT and PVP; Pathway two: directly inducing adventitious roots from leaves Leaves obtained in step 3) are cut and several incisions are made on the leaves, and the leaves are inoculated in an adventitious root direct induction medium to induce adventitious roots; The adventitious root direct induction medium comprises IBA, NAA, KT and PVP; Pathway three: directly inducing adventitious roots from stems Stems obtained in step 3) are cut and inoculated in an adventitious root direct induction medium to induce adventitious roots; The adventitious root direct induction medium comprises IBA, NAA, KT and PVP.

2. The method for inducing camphor tree adventitious roots according to claim 1, characterized in that, Step 1) The establishment of the sterile system of Cinnamomum camphora comprises: selecting tender shoots sprouted this year as explants, sequentially immersing the explants in 0.2% sodium hypochlorite solution for sterilization for 10-15 min, immersing the explants in 0.1% mercuric chloride solution for sterilization for 5-10 min, and sufficiently rinsing the explants with sterile water.

3. The method for inducing camphor tree adventitious roots according to claim 1, characterized in that, Step 2) The shoot induction medium comprises: MS solid medium, and 0.5-2.0 mg / L 6-BA and 0.05-0.5 mg / L NAA are added.

4. The method of inducing camphor tree adventitious roots according to claim 1, characterized in that, Step 3) The sterile seedling medium comprises: MS solid medium, and 0.5-2.0 mg / L 6-BA, 0.05-0.5 mg / L NAA, 0.1-0.5 mg / L GA3 and 0.05%-0.2% (w / v) activated carbon are added.

5. The method of inducing camphor tree adventitious roots according to claim 1, characterized in that, In pathway one of step 4), the callus induction medium comprises: B5 solid medium, and 0.5-2 mg / L 2,4-D, 0.1-1 mg / L 6-BA, 0.1-0.3 mg / L TDZ, 100-500 mg / L Pro and 1-2 g / L PVP are added.

6. The method of inducing camphor tree adventitious roots according to claim 1, wherein, In pathway one of step 4), the adventitious root induction medium comprises: 1 / 2MS solid medium, and 2-6 mg / L IBA, 0.1-0.5 mg / L NAA, 0.1-0.3 mg / L KT and 1-2 g / L PVP are added.

7. The method of inducing camphor tree adventitious roots according to claim 1, wherein, Step 4) In the second approach, the direct induction medium for adventitious roots comprises 1 / 2MS solid medium supplemented with 0.5-2.5 mg / L IBA, 0.1-0.5 mg / L NAA, 0.1-0.3 mg / L KT and 1-2 g / L PVP.

8. The method of inducing camphor tree adventitious roots according to claim 1, wherein, Step 4) In the third approach, the direct induction medium for adventitious roots comprises 1 / 2MS solid medium supplemented with 0.5-2.5 mg / L IBA, 0.1-0.5 mg / L NAA, 0.1-0.3 mg / L KT and 1-2 g / L PVP.

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