In-vitro rapid propagation method for phoebe bournei stems

Abstract

The invention discloses a phoebe bournei stem in-vitro rapid propagation method, and relates to the technical field of forest tree tissue culture.The phoebe bournei semi-wood branch is used as an explant, two reagents are used for double-effect sterilization, and a sterile material is rapidly and efficiently obtained; plant growth regulators with different component combinations and concentrations are added for systematic research on axillary bud induction culture, adventitious bud subculture multiplication culture and rooting culture, and browning prevention and control and strong seedling culture tests are further carried out aiming at the browning phenomenon of phoebe bournei cluster buds in the adventitious bud subculture multiplication culture process; according to the method, effective measures for preventing phoebe bournei subculture browning and a method for improving seedling quality are screened, a phoebe bournei stem in-vitro rapid propagation technical system is successfully constructed, a reliable method is provided for phoebe bournei precious resource preservation and large-scale propagation, and a technical reference is provided for in-vitro culture research of other phoebe tree species.

Description

A method for rapid in vitro propagation of *Phoebe zhennan* stem segments Technical Field

[0001] This invention relates to the field of forest tree tissue culture technology, and in particular to a method for rapid in vitro propagation of Phoebe zhennan stem segments. Background Technology

[0002] Phoebe bournei, an evergreen tree belonging to the genus Phoebe in the family Lauraceae, is a rare and precious tree species endemic to my country. Its wood is of excellent quality, easy to process, resistant to insects and pests, and has good paint-making properties, making it a superior material for high-end furniture, fine construction, craft carving, and shipbuilding. The oil extracted from its fruit can be used to produce biodiesel. Furthermore, Phoebe bournei is also an excellent landscaping tree due to its tall stature, thick canopy, dense foliage, and evergreen nature.

[0003] Due to its high economic value, *Phoebe zhennan* has been over-logged, and its suitable growing environment has been destroyed by human activities, resulting in a significant reduction in natural forest resources. To address the protection and cultivation of *Phoebe zhennan*, researchers have conducted a series of technical studies. For example, regarding the mixed forest model of *Phoebe zhennan*, the article "The Influence of Mixing Ratio on the Growth Morphology and Space Utilization Capacity of *Phoebe zhennan*-Chinese Cunninghamia lanceolata* Artificial Mixed Forest" by Zhou Xinhua, Sun Yun, Wang Liyun, et al., shows that mixed treatment promotes the growth of *Phoebe zhennan* and can also improve its space utilization capacity, thus having a positive effect on the protection of *Phoebe zhennan*. Regarding the asexual reproduction of *Phoebe zhennan*, many studies have been conducted on its rapid propagation by cuttings, for example, by Chen Minggao, Wu Jiyou, Shu Yao, et al. The article "Experiment on Asexual Propagation of Machilus chinensis by Cuttings" [J] conducted a rooting experiment on tender branches of Machilus chinensis clonal cuttings in a nursery to explore the rooting pattern and rooting ability of Machilus chinensis clonal cuttings, providing a theoretical basis for the rapid propagation of Machilus chinensis seedlings. However, cutting propagation is limited by factors such as mother plant, season, and difficulty in virus removal. Long-term propagation can lead to seedling degeneration, reduced yield and quality.

[0004] Asexual reproduction through tissue culture can overcome the above problems. However, there are few research reports on rapid propagation technology of *Machilus chinensis* through tissue culture. Publication number CN104719155A discloses a method for rapid propagation of *Machilus chinensis* through tissue culture. It discloses that by using stem segments with buds as explants, and through processes such as induction culture, subculture, rooting, hardening and transplanting, *Machilus chinensis* regenerated plants were successfully obtained in vitro. However, this study only conducted a preliminary exploration of tissue culture of stem segments with buds. In the established rapid in vitro propagation technology system of *Machilus chinensis*, the proliferation rate of axillary buds was low, only between 37-45%. The rooting rate of the buds after rooting culture was between 73.7-78%, and the survival rate of the transplanted test-tube seedlings was between 76.9-81.3%, indicating room for improvement.

[0005] The core technical bottleneck in woody plant tissue culture lies in the selection and sterilization of explants. Problems also include difficulty in initial induction, low differentiation rates, low subculture proliferation coefficients, difficulty in rooting, and low transplant survival rates. Furthermore, woody plants contain high levels of phenolic substances and have high oxidase activity, leading to browning during in vitro culture, which severely affects proliferation efficiency. These key technical points present significant bottlenecks in both research and production, hindering the establishment of a complete in vitro culture technology system.

[0006] This invention addresses the aforementioned technical bottlenecks by starting with low-damage, high-sterilization explant disinfection techniques. Through the scientific combination of basal culture medium and growth regulators, it enhances the differentiation efficiency of initially induced buds, the proliferation coefficient of subcultures, and the number of roots in rooting culture. Simultaneously, the addition of antioxidants and adsorbents to subcultures inhibits browning, overcoming technical difficulties at each stage. Ultimately, this invention establishes a rapid and efficient in vitro regeneration method for *Machilus chinensis*, providing technical support for the large-scale production of *Machilus chinensis*. This method also serves as a foundational platform for innovation in *Machilus chinensis* breeding technology and is of great significance for the protection and development of *Machilus chinensis* resources. Summary of the Invention

[0007] The purpose of this invention is to propose a rapid in vitro propagation method for *Phoebe zhennan* stem segments to overcome the above-mentioned technical problems, providing a reliable method for the preservation and large-scale propagation of precious *Phoebe zhennan* resources, and providing a reference for in vitro culture research of other *Phoebe* species.

[0008] The present invention is achieved through the following technical solution: The present invention provides a method for rapid in vitro propagation of Phoebe bournei stem segments, including the following steps: (1) Sterilization treatment of explants: Select healthy, disease-free semi-lignified stem segments from a single Phoebe bournei plant as explants, and after pruning and sterilization treatment, they are ready for use; (2) Axillary bud induction culture: Cut the explants obtained by pretreatment in step (1) into segments with 1-2 buds, and inoculate them into the induction culture medium for induction culture; (3) Adventitious bud subculture culture: Cut the axillary buds that have sprouted 2-3 fully expanded leaves and grown to 2cm from the stem segment from the induction culture medium, and inoculate them into the adventitious bud subculture culture medium for adventitious bud subculture culture. The adventitious bud subculture culture medium is MS as the base medium, with 0.2-1 added. 0 mg / L 6-benzylaminopurine, 0.01-0.1 mg / L thiamethoxam and 0.1-0.5 mg / L indolebutyric acid; while the adventitious buds were subcultured and propagated, anti-browning seedling treatment was carried out. The anti-browning seedling treatment was based on the components of the adventitious bud subculture and propagation medium, with the addition of 0.1 mg / L adenosine monophosphate and 0.2 g / L activated carbon; (4) Rooting culture, hardening and transplanting: select healthy single adventitious buds of Phoebe zhennan obtained by adventitious bud subculture and propagation, inoculate them into the rooting medium for rooting culture, obtain rooted tissue culture seedlings, and then harden and transplant the rooted tissue culture seedlings; the rooting medium is based on 1 / 2 MS medium, with the addition of 0.1-0.2 mg / L naphthaleneacetic acid and 0.1-0.2 mg / L activated carbon.

[0009] As a further optimization of the present invention, in step (1), the pruning and sterilization treatment steps are as follows: after removing the leaves and surface dirt from the explant, rinse it under running water for 2 hours. Then, treat the stem segment with 75% alcohol for 45 seconds, then treat the stem segment with 2% sodium hypochlorite for 10 minutes, then treat the stem segment with 0.1% mercuric chloride reagent for 6 minutes, and finally rinse the stem segment with sterile water 3-4 times.

[0010] As a further optimization of the present invention, in step (2), the induction culture medium is MS + 1.0 mg / L 6-benzylaminopurine + 0.5 mg / L indolebutyric acid.

[0011] As a further optimization of the present invention, in step (3), the adventitious bud subculture proliferation medium is based on MS medium, with 0.2 mg / L 6-benzylaminopurine, 0.05 mg / L thiamethoxam and 0.5 mg / L indolebutyric acid added.

[0012] As a further optimization of the present invention, the induction medium, the adventitious shoot subculture medium and the rooting medium are further supplemented with 30 g / L sucrose and 6.5 g / L carrageenan, and the pH value of the induction medium and the adventitious shoot subculture medium is controlled at 5.8-6.0.

[0013] As a further optimization of the present invention, in steps (2) to (4), the culture conditions for axillary bud induction culture, adventitious bud subculture and rooting culture are: temperature 25±2℃, light intensity 2500Lux, white light, and light duration 14h / d.

[0014] As a further optimization of the present invention, the anti-browning seedling treatment cycle is 120 days, and the culture medium is replaced once every 40 days.

[0015] As a further optimization of the present invention, in step (4), the specific steps of seedling hardening and transplanting are as follows: first, place the rooted tissue culture seedlings with bottles on the greenhouse seedling rack for hardening for 7 days, then take out the rooted tissue culture seedlings, wash off the culture medium, and transplant them into container bags.

[0016] As a further optimization of the present invention, the transplanting substrate filled in the container bag comprises, by weight percentage, 75% peat moss, 10% perlite, 10% coconut coir, and 5% wood ash.

[0017] Compared with the prior art, the present invention has the following advantages: (1) The present invention uses semi-woody branches of Phoebe bournei as explants. When establishing an aseptic system for explants, the innovative disinfection mode reduces the damage to explant materials caused by excessively long mercuric chloride disinfection and the situation of incomplete disinfection, thereby increasing the survival rate of axillary buds in stem segments to 51.67% and reducing the contamination rate to 38.33%; (2) The present invention introduces browning prevention and seedling strengthening treatment in the process of adventitious bud subculture. Studies have found that adding adenosine monophosphate and activated carbon to the adventitious bud subculture culture medium has a significant anti-browning effect, and is also conducive to the high growth of clustered buds, the elongation and thickening of stems, and the color of leaves. The leaves change from light green to dark green, and from thin to thick and from narrow to wide, which is conducive to plant photosynthesis and helps prevent browning in subsequent rooting culture; (3) In the rooting culture stage, this invention can induce robust roots by using only a single growth hormone, and the rooting rate can reach more than 90%. The operation is simple, direct and efficient, which helps to improve the survival rate of transplanted rooted tissue culture seedlings. In the end, the survival rate of transplanted seedlings can be increased to more than 90%; In summary, this invention provides a reliable method for the preservation and large-scale propagation of precious resources of Phoebe zhennan by constructing a rapid propagation technology system for Phoebe zhennan stem segments, and provides a reference for the in vitro culture research of other Phoebe species. Attached Figure Description

[0018] Figure 1 is a schematic diagram of the collection and processing of experimental materials provided by the present invention; in the figure, A is the current year semi-lignified branch of *Machilus pekinensis*; B is the axillary bud germination state of explants inducing culture medium A-1 for 10 days; C is the axillary bud germination state of explants inducing culture medium A-1 for 20 days; Figure 2 shows the results of axillary bud induction of *Machilus pekinensis* explants by different basal culture media provided by the present invention (in the figure, from left to right, they correspond to groups B-1, B-2, B-3, B-4, and B-5); Figure 3 shows the state of adventitious bud clusters in the early stage of induction of representative treatment groups C-7, C-8, C-9, C-13, and C-16 selected by the present invention; Figure 4 shows the adventitious bud proliferation and subculturing three times in practice provided by the present invention. The adventitious buds that later showed browning; Figure 5 shows the effect of different antioxidants provided by the present invention on browning and seedling vigor during the propagation of *Machilus pekinensis* (in the figure, from left to right, they correspond to groups D-1, D-2, D-3, D-4, and D-5); Figure 6 shows the comparison between the D-4 treatment group provided by the present invention and the clustered buds without anti-browning seedling vigor treatment; Figure 7 shows the effect of different auxin combinations provided by the present invention on the rooting of *Machilus pekinensis* tissue culture seedlings (in the figure, from left to right and from top to bottom, they correspond to groups E-2, E-3, E-4, E-5, E-6, E-7, E-8, and E-9); Figure 8 shows the rooting effect of *Machilus pekinensis* tissue culture seedlings in the E-4 treatment group provided by the present invention; Figure 9 shows the transplant survival rate of *Machilus pekinensis* tissue culture rooted seedlings obtained in the E-4 treatment group provided by the present invention. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0020] Unless otherwise specified, all reagents and materials used below are commercially available products.

[0021] The culture media used in each of the following culture stages were also supplemented with 30 g / L sucrose and 6.5 g / L carrageenan, and the pH of the culture media used in each culture stage was controlled at 5.8-6.0.

[0022] II. Methods Unless otherwise specified, all methods used below are conventional methods known to those skilled in the art.

[0023] Experimental materials were collected from the *Machilus macrantha* gene bank of the Jiangxi Academy of Forestry Sciences (27°53′N, 116°47′E). In mid-April to early May 2024, on sunny mornings from 9:00 to 10:00, healthy, disease-free semi-lignified branches of the current year from individual *Machilus macrantha* trees were collected (Figure A in Figure 1) as explants.

[0024] To construct a sterile system, leaves were removed from the explants, and the branches were cleaned with detergent to remove surface dirt, then rinsed under running water for 2 hours. After surface disinfection with 75% alcohol for 45 seconds on a laminar flow hood, the explants were then disinfected for different durations using 2% sodium hypochlorite and 0.1% mercuric chloride reagents, respectively. After rinsing 3-4 times with sterile water to remove residual disinfectant, the explants were cut into stem segments with a single axillary bud and inoculated into induction medium (MS + 1.0 mg / L 6-benzylaminopurine 6-BA + 0.5 mg / L indolebutyrate IBA) for axillary bud induction culture experiments to investigate the effect of different disinfection times on the growth of *Machilus macrantha* axillary bud stem segments. Each treatment was inoculated with 20 stem segments with buds, and each treatment was replicated in triplicate.

[0025] Subsequently, under the cultivation conditions of (25±2)℃, light intensity of 2500lx, and light duration of 14h / d, after 20 days of cultivation, the number of contaminated axillary bud stem segments and the number of surviving axillary bud stem segments were counted, and the axillary bud contamination rate and axillary bud survival rate were calculated according to the following formulas: Axillary bud contamination rate = (number of contaminated axillary bud stem segments / number of inoculated stem segments) × 100%; Axillary bud survival rate = (number of surviving axillary bud stem segments / number of inoculated stem segments) × 100%; The method for judging the contamination of axillary bud stem segments is: the formation of fungal or bacterial infection around the axillary bud stem segment inserted into the culture medium (fungal infection: white, green, black or gray fluffy or flocculent small spots appear on the surface of the culture medium; bacterial infection: water-soaked, transparent or translucent small colonies appear on the surface of the culture medium, which are mucous or purulent).

[0026] The results are shown in Table 1.

[0027] Table 1. Effects of different disinfection times on the growth of axillary buds and stem segments of *Machilus macrantha*. Note: The data in the table are "mean ± standard deviation". Different lowercase letters after the data in the same column indicate significant differences (P<0.05). The same applies to the following tables.

[0028] As can be seen from Table 1, sterilization with 2% NaClO alone for 20 minutes resulted in the highest contamination rate, while sterilization with 0.1% HgCl2 alone for 10 minutes resulted in browning at the explant incision site and the lowest survival rate of only 44.8%. This indicates that a single disinfectant cannot simultaneously achieve both low contamination rate and high survival rate.

[0029] The experiment found that applying two disinfectants simultaneously could effectively reduce the contamination rate and help improve the survival rate of axillary buds. When the NaClO treatment time was the same, the contamination rate and survival rate both showed a decreasing trend as the HgCl2 sterilization time was extended. When 2% NaClO was treated for 10 min and 0.1% HgCl2 was treated for 6 min, the contamination rate was significantly lower than other treatment combinations (P<0.05), and the survival rate was not significantly different compared to other treatments.

[0030] Therefore, the optimal disinfection conditions for the axillary bud stem segments of *Phoebe zhennan* are: first, treat with 2% NaClO for 10 min, then treat with 0.1% HgCl2 for 6 min (treatment group A-6).

[0031] 3. Axillary bud induction culture: 1.0 mg / L 6-benzylaminopurine (6-BA) and 0.5 mg / L indoleacetic acid (IBA) were added to five basal culture media, namely MS, WPM, B5, N6, and White media (all using Solarbio). Twenty stem segments with 1-2 buds were inoculated for each treatment (sterilized according to the sterilization conditions of treatment group A-6 before inoculation), and each treatment was set up in 3 replicates. The axillary bud initiation time was recorded. After culturing for 20 days under the conditions of (25±2)℃, light intensity of 2500 lx, and light duration of 14 h / d, the axillary bud induction rate and growth status were recorded.

[0032] The axillary bud induction rate is calculated using the following formula: Axillary bud induction rate = (Number of stem segments that induced axillary buds / Number of inoculated stem segments) × 100%.

[0033] The results are shown in Table 2.

[0034] Table 2. Effects of different basal culture media on axillary bud induction in *Machilus pekinensis* explants. As shown in Table 2, the axillary bud induction rate of the stem segments with buds in MS medium of treatment group B-1 was significantly higher than that of other basal media (P<0.05), while there was no significant difference in the axillary bud induction rate of the remaining four basal media.

[0035] Ten days after inoculation, the axillary buds of treatment group B-1 began to swell and develop grayish-white bud tips. Over time, smooth, tender green leaves appeared (Figure B in Figure 1). Twenty days after inoculation, they developed into complete tender branches, which can be used as excellent sterile materials for subsequent culture (Figure C in Figure 1).

[0036] Figure 2 shows the results of axillary bud induction in different culture media for *Machilus pekinensis* explants (from left to right, these correspond to groups B-1, B-2, B-3, B-4, and B-5). In group B-1, the axillary buds in MS medium developed smooth, tender green leaves. In groups B-2, B-3, WPM, and B-5, the axillary buds were small and the leaves were yellowish. In group B-4, the axillary buds in N6 medium germinated and the leaves were yellowish and vitrified. In group B-5, the axillary buds in White medium had a long germination period and the edges of the germinated leaves were yellowish.

[0037] 4. Adventitious bud subculture: From treatment group B-1, select axillary buds that have sprouted 2-3 fully expanded leaves and grown to 2cm, cut them off from the stem segment, and inoculate them into adventitious bud subculture.

[0038] To investigate the effects of different compositions of adventitious shoot subculture media on adventitious shoot proliferation, MS medium was used as the basal medium, with different concentrations of 6-benzylaminopurine (6-BA) (0, 0.2, 0.5, 1.0 mg / L), thidiazuron (TDZ) (0, 0.01, 0.05, 0.1 mg / L), and indolebutyric acid (IBA) (0, 0.1, 0.2, 0.5 mg / L). Five bottles were placed in each treatment, with two axillary shoots in each bottle, and four replicates were set for each treatment. After one cycle (40 days) of culture at a temperature of (25±2)℃, a light intensity of 2500 lx, and a photoperiod of 14 h / d, the proliferation coefficient was calculated and the proliferation and growth of adventitious shoots were observed.

[0039] The proliferation coefficient was calculated using the following formula: Proliferation coefficient = Number of shoots after one culture cycle / Number of inoculated shoots; the results are shown in Table 3.

[0040] Table 3. Effects of different concentrations of culture medium and growth regulator combinations on adventitious shoot proliferation. Note: k1, k2, k3 and k4 represent the mean values ​​of the adventitious bud proliferation coefficients at the four levels of each factor, and R represents the range.

[0041] As shown in Table 3, after cutting off the axillary buds that germinated in the induction medium and inoculating them into the subculture proliferation medium, the effects of different concentrations of plant growth regulator combinations on the adventitious bud proliferation coefficient were significantly different (P<0.05).

[0042] Range analysis showed that the effects of different plant growth regulators on the proliferation of adventitious buds of *Machilus macrantha* were represented by R0. 6-BA >R TDZ >R IBAThe results indicate that 6-BA has the greatest impact on the proliferation of adventitious buds in *Machilus macrantha*. The proliferation coefficient of adventitious buds increases with increasing 6-BA concentration, but after treatment with high concentrations of 6-BA, the plants differentiated a large number of tender green adventitious buds, and the leaves were pale green and transparent with a water-soaked appearance, which was not suitable for subsequent cultivation. Under the action of higher concentrations of the cytokinin 6-BA and TDZ, the buds of *Machilus macrantha* grew in a "ginger"-like sheet-like manner, with strong bud differentiation ability, clear basal callus, and small leaves (C-13 treatment group). Figure 3 shows the adventitious bud clusters in the early stage of induction of several representative treatment groups, corresponding to treatment groups C-7, C-8, C-9, C-13, and C-16, respectively.

[0043] In summary, the C-7 treatment group represents the optimal culture medium conditions for the proliferation of adventitious buds of *Machilus macrantha*, namely MS + 0.2 mg / L 6-BA + 0.05 mg / L TDZ + 0.5 mg / L IBA.

[0044] 5. Browning prevention and seedling cultivation: The adventitious buds of Phoebe bournei are all in a clustered state (adventitious bud cluster), the buds are dense and short, and the leaves are clearly wrapped around the stem, which is not conducive to rooting in the later stage. Moreover, with the increase of subculture, more and more metabolic products accumulate around the inserted culture medium, the culture medium turns black, and brown spots appear on the bud leaves, which seriously inhibits the growth of propagation bottle seedlings. Figure 4 shows the adventitious buds that showed browning after three subcultures in practice.

[0045] Therefore, in the process of adventitious shoot subculture, different concentrations of anti-browning agents (vitamin C (VC), polyvinylpyrrolidone (PVP), silver nitrate (AgNO3), adenosine monophosphate (AMP), cysteine ​​(Cys)) and adsorbent-activated carbon (AC) were added to the subculture medium (treatment group C-7) selected above to prevent browning and promote seedling growth. Five seedlings were inoculated for each treatment, and three replicates were set for each treatment.

[0046] After culturing for 120 days at a temperature of (25±2)℃, a light intensity of 2500lx, and a light duration of 14h / d, the culture medium was changed every 40 days, and the browning rate of adventitious buds, growth status, and width of the first leaf were recorded.

[0047] The results are shown in Table 4.

[0048] Table 4. Effects of different antioxidants on browning and seedling vigor during the propagation of Machilus thunbergii. As shown in Table 4 and Figure 5, the addition of adenosine monophosphate (AMP) and activated carbon (AC) to the culture medium has a significant anti-browning effect and is also beneficial to the high growth of clustered buds, with stems becoming longer and thicker, and the color of leaves changing from light green to dark green, and the leaves becoming thicker and wider. According to the statistical results, the average leaf width of the D-4 treatment group reached 1.19 cm, the leaf surface was glossy, and the number of unfolded leaves increased significantly. The average leaf width of the other antioxidants added was less than 1 cm, the browning rate was higher than 50%, and there were no significant changes in the seedlings (Figure 6 is a comparison between the D-4 treatment group and the seedlings without anti-browning treatment).

[0049] In summary, adding 0.1 mg / L adenosine monophosphate (AMP) + 0.2 g / L activated carbon (AC) to each liter of subculture proliferation medium resulted in the best number of effective seedlings and seedling growth. Therefore, the optimal subculture proliferation medium for preventing browning and strengthening seedlings of *Machilus chinensis* is MS + 0.2 mg / L 6-BA + 0.01 mg / L TDZ + 0.5 mg / L IBA + 0.1 mg / L AMP + 0.2 g / L AC.

[0050] 6. Rooting Culture and Seedling Hardening Transplanting: Vigorous adventitious buds from treatment group D-4 were selected and transferred to rooting medium for rooting culture. Different concentrations of NAA (0, 0.1, 0.2 mg / L), indoleacetic acid (IBA) (0, 0.5, 1.0 mg / L), and activated carbon AC (0, 0.1, 0.2 mg / L) were added to 1 / 2 MS medium. Five bottles were placed in each treatment, with 10 adventitious buds in each bottle, and each treatment was replicated three times. After 15 days of culture, the rooting rate and rooting status were recorded.

[0051] Subsequently, the rooted tissue culture seedlings were placed on the greenhouse seedling rack for 7 days to harden off. The rooted seedlings were then removed, the culture medium was washed off, and they were transplanted into 6cm×8cm container bags. The main components of the substrate in the container bags were 75% peat moss, 10% perlite, 10% coconut coir, and 5% wood ash. After transplanting, the seedlings were covered with a film to keep them moist, and the film was removed depending on the weather. The seedling growth was observed and the survival rate was recorded one month later.

[0052] The rooting rate and survival rate were calculated using the following formulas: Rooting rate = (Number of roots / Number of inoculated single buds) × 100%; Transplant survival rate = (Number of surviving plants / Total number of transplanted plants) × 100%; The results are shown in Table 5.

[0053] Table 5. Effects of different auxin combinations on rooting and transplant survival of *Machilus chinensis* tissue culture seedlings. Note: k1, k2, and k3 represent the mean rooting rates of the three levels for each factor, and R represents the range.

[0054] As can be seen from Table 5, Figure 7 and Figure 8, the rooting rate of seedlings under different combinations of conditions differed significantly (p<0.05). In 1 / 2 MS basal medium without growth regulators, the stem buds could not grow roots. In the single exogenous auxin combination, the adventitious buds treated with NAA had significantly higher rooting rate and average number of roots than those treated with IBA. At the same time, in the combination of applying two exogenous auxins, the normal growth of the root system was hindered, and the adventitious roots showed swollen and deformed phenomena.

[0055] Under the E-4 combination (1 / 2MS + 0.1 mg / L NAA + 0.1 g / L AC), the rooting time was the shortest, with white root primordia appearing as early as 10 days. Each plant had up to 6 fleshy and robust roots, and the combination of naphthaleneacetic acid and activated carbon could induce root growth. However, the two growth regulators (NAA and IBA) were actually detrimental to rooting, with silvery-white nodules appearing on the roots and the roots becoming curled. This may be due to the multiple effects of the growth regulators hindering root development and the appearance of abnormally enlarged parts.

[0056] During root induction, activated carbon inhibited browning. By increasing the concentration of activated carbon, the adventitious roots changed from brown to white, and the browning at the base of the adventitious buds was alleviated, resulting in whiter roots than those without activated carbon. Considering both the quantity and quality of roots, the optimal culture medium for transplanting *Machilus chinensis* tissue culture seedlings was 1 / 2 MS + 0.1 mg / L NAA + 0.1 g / L AC. Finally, the transplant survival rate of *Machilus chinensis* tissue culture seedlings obtained from the E-4 treatment group is shown in Figure 9.

[0057] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the protection scope of the present invention.

Claims

1. A method for rapid in vitro propagation of *Phoebe zhennan* stem segments, characterized in that, Includes the following steps: (1) Sterilization of explants: Select healthy, disease-free semi-lignified stem segments from the current year of a single *Machilus chinensis* plant as explants. After pruning and sterilization, they are ready for use. (2) Axillary bud induction culture: Cut the explants obtained in step (1) into segments with 1-2 buds each, and inoculate them into the induction medium for induction culture. (3) Adventitious bud subculture: Cut axillary buds that have sprouted 2-3 fully expanded leaves and reached 2 cm in length from the stem segments in the induction medium and inoculate them into the adventitious bud subculture culture medium for adventitious bud subculture culture. The adventitious bud subculture culture medium is based on MS medium, with 0.2-1.0 mg / L 6-benzylaminopurine and 0.01-0.1 mg / L 6-benzylaminopurine added. mg / L thiamethoxam and 0.1-0.5 mg / L indolebutyric acid; while the adventitious buds were subcultured and propagated, anti-browning seedling treatment was carried out. The anti-browning seedling treatment was based on the components of the adventitious bud subculture and propagation medium, with the addition of 0.1 mg / L adenosine monophosphate and 0.2 g / L activated carbon; (4) Rooting culture, hardening and transplanting: select healthy single adventitious buds of Phoebe bournei obtained by adventitious bud subculture and propagation, inoculate them into the rooting medium for rooting culture, obtain rooted tissue culture seedlings, and then harden and transplant the rooted tissue culture seedlings; the rooting medium is based on 1 / 2 MS medium, with the addition of 0.1-0.2 mg / L naphthaleneacetic acid and 0.1-0.2 mg / L activated carbon.

2. The method for rapid in vitro propagation of *Machilus chinensis* stem segments according to claim 1, characterized in that, In step (1), the pruning and sterilization process is as follows: After removing the leaves and surface dirt from the explant, rinse it under running water for 2 hours. Then, treat the stem segment with 75% alcohol for 45 seconds, then treat the stem segment with 2% sodium hypochlorite for 10 minutes, then treat the stem segment with 0.1% mercuric chloride reagent for 6 minutes, and finally rinse the stem segment with sterile water 3-4 times.

3. The method for rapid in vitro propagation of *Phoebe zhennan* stem segments according to claim 1, characterized in that, In step (2), the induction medium is MS + 1.0 mg / L 6-benzylaminopurine + 0.5 mg / L indolebutyric acid.

4. The method for rapid in vitro propagation of *Machilus chinensis* stem segments according to claim 1, characterized in that, In step (3), the adventitious shoot subculture culture medium is based on MS medium, with 0.2 mg / L 6-benzylaminopurine, 0.05 mg / L thiamethoxam and 0.5 mg / L indolebutyric acid added.

5. A method for rapid in vitro propagation of *Phoebe zhennan* stem segments according to any one of claims 1-4, characterized in that, The induction medium, adventitious bud subculture medium, and rooting medium are further supplemented with 30 g / L sucrose and 6.5 g / L carrageenan, and the pH values ​​of the induction medium, adventitious bud subculture medium, and rooting medium are all controlled at 5.8-6.

0.

6. The method for rapid in vitro propagation of *Machilus chinensis* stem segments according to claim 1, characterized in that, In steps (2) to (4), the culture conditions for axillary bud induction culture, adventitious bud subculture and rooting culture are: temperature 25±2℃, light intensity 2500Lux, white light, and light duration 14h / d.

7. The method for rapid propagation of *Machilus chinensis* stem segments in vitro according to claim 1, characterized in that, The treatment cycle for preventing browning and strengthening seedlings was 120 days, with the culture medium being replaced every 40 days.

8. The method for rapid in vitro propagation of *Machilus chinensis* stem segments according to claim 1, characterized in that, In step (4), the specific steps for hardening and transplanting seedlings are as follows: first, place the rooted tissue culture seedlings with bottles on the greenhouse seedling rack for hardening for 7 days, then take out the rooted tissue culture seedlings, wash off the culture medium, and transplant them into container bags.

9. The method for rapid in vitro propagation of *Machilus chinensis* stem segments according to claim 8, characterized in that, The transplanting substrate filled in the container bag comprises, by weight percentage, 75% peat moss, 10% perlite, 10% coconut coir, and 5% wood ash.

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