In vitro rapid propagation method of phoebe sheareri stem segments

Abstract

This invention discloses a rapid in vitro propagation method for *Phoebe zhennan* stem segments, belonging to the field of forest tree tissue culture technology. Using semi-woody branches of *Phoebe zhennan* as explants, and employing two reagents for double-effect sterilization, sterile materials are rapidly and efficiently obtained. Then, by adding plant growth regulators of different combinations and concentrations, axillary bud induction culture, adventitious bud subculture, and rooting culture are systematically studied. Furthermore, regarding the browning phenomenon occurring in clustered buds of *Phoebe zhennan* during adventitious bud subculture, browning prevention and seedling strengthening experiments are conducted to screen effective measures to prevent browning in *Phoebe zhennan* subculture and methods to improve seedling quality. A rapid in vitro propagation technology system for *Phoebe zhennan* stem segments has been successfully constructed, providing a reliable method for the preservation and large-scale propagation of this precious resource, and offering technical reference for in vitro culture research of other *Phoebe* species.

Description

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] This invention is achieved through the following technical solution: This invention provides a method for rapid in vitro propagation of *Phoebe zhennan* stem segments, comprising the following steps: (1) Sterilization treatment of explants: Select healthy, disease-free semi-lignified stem segments from the current year of a single Phoebe bournei plant as explants, and after pruning and sterilization treatment, they are ready for use; (2) Axillary bud induction culture: The explants obtained by pretreatment in step (1) are cut into small segments of 1-2 buds and inoculated into the induction culture medium for induction culture; (3) Adventitious bud subculture: Axillary buds that have sprouted 2-3 fully expanded leaves and grown to 2 cm in length are cut from the stem segment from the induction medium and inoculated into the adventitious bud subculture medium for adventitious bud subculture. The adventitious bud subculture medium is MS as the basal medium, with 0.2-1.0 mg / L 6-benzylaminopurine, 0.01-0.1 mg / L thiamethoxam and 0.1-0.5 mg / L indolebutyric acid added. During the adventitious bud subculture, a browning prevention and seedling strengthening treatment was performed. The browning prevention and seedling strengthening treatment was performed by adding 0.1 mg / L adenosine monophosphate and 0.2 g / L activated carbon to the adventitious bud subculture culture medium. (4) Rooting culture, hardening and transplanting: Select healthy single adventitious buds of Phoebe bournei obtained by adventitious bud subculture and inoculate them into rooting culture medium to obtain rooted tissue culture seedlings. Then, harden and transplant the rooted tissue culture seedlings. The rooting culture medium is based on 1 / 2MS medium with 0.1-0.2 mg / L naphthaleneacetic acid and 0.1-0.2 mg / L activated carbon added.

[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] The present invention has the following advantages over the prior art: (1) This 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 stems grow longer and thicker, the color of the leaves changes from light green to dark green, the leaves change from thin to thick, and from narrow to wide, which is conducive to plant photosynthesis and helps to prevent browning in subsequent rooting culture. (3) In the rooting culture stage, the present 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. The final transplanting survival rate can be increased to more than 90%. In summary, this invention provides a reliable method for the preservation and large-scale propagation of precious Phoebe zhennan resources by constructing an in vitro rapid propagation technology system, and provides a reference for in vitro culture research of other Phoebe species. Attached Figure Description

[0018] Figure 1 The diagram shows the collection and processing of experimental materials provided by this invention; in the diagram, A is a semi-lignified branch of the current year of the *Phoebe zhennan*; B is a diagram of the axillary bud germination state after explants are inoculated into treatment group A-1 induction medium and cultured for 10 days; C is a diagram of the axillary bud germination state after explants are inoculated into treatment group A-1 induction medium and cultured for 20 days. Figure 2 The results of different basal culture media provided by this invention on the induction of axillary buds of Machilus pekinensis explants (in the figure, from left to right, they correspond to group B-1, group B-2, group B-3, group B-4 and group B-5). Figure 3 The diagram shows the state of adventitious bud clusters in the early stage of induction for the representative C-7, C-8, C-9, C-13 and C-16 treatment groups selected for this invention. Figure 4 The adventitious buds that show browning after three subcultures during the practice provided by this invention; Figure 5The effects of different antioxidants provided by this invention on browning and seedling vigor during the propagation of *Machilus macrantha* (in the figure, from left to right, they correspond to groups D-1, D-2, D-3, D-4, and D-5). Figure 6 A comparison of the D-4 treatment group provided by this invention with the clustered shoots that have not undergone anti-browning seedling treatment; Figure 7 The effects of different combinations of auxin substances provided by this invention on the rooting of *Machilus chinensis* 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 The rooting effect of the E-4 treatment group of *Phoebe zhennan* tissue culture seedlings provided by this invention; Figure 9 The transplant survival rate of *Phoebe zhennan* tissue culture rooted seedlings obtained from the E-4 treatment group provided by this 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] Reagents and materials 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, the methods used below are conventional methods known to those skilled in the art.

[0023] Test materials The experimental materials were collected from the *Machilus macrantha* gene resource bank of the Jiangxi Academy of Forestry Sciences (27°53′N, 116°47′E). Healthy, disease-free semi-lignified branches from the current year were collected from individual *Machilus macrantha* trees between 9:00 and 10:00 AM on sunny mornings from mid-April to early May 2024. Figure 1 (Figure A in the diagram), used as an explant.

[0024] Building 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 clean bench, 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 indolebutyric acid IBA) for axillary bud induction culture experiments to investigate the effect of different disinfection times on the growth of *Machilus chinensis* 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 determining whether axillary bud stem segments are contaminated is as follows: fungal or bacterial infection forms around the culture medium when the axillary bud stem segment is inserted (fungal infection: white, green, black or gray fluffy or flocculent dots 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: MS, WPM, B5, N6, and White (all using Solarbio). Twenty stem segments with 1-2 buds were inoculated into each treatment (sterilized according to the sterilization conditions of treatment group A-6 before inoculation), and each treatment was replicated three times. The axillary bud initiation time was recorded. After culturing for 20 days at a culture temperature of (25±2)℃, a light intensity of 2500 lx, and a photoperiod 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, eventually producing smooth, tender green leaves over time. Figure 1 (See Figure B in the image). After 20 days of inoculation, the shoots developed into complete tender branches, which can be used as excellent sterile materials for subsequent culture. Figure 1 (Figure C in the diagram).

[0036] Figure 2The results of axillary bud induction in different culture media for *Machilus chinensis* explants are shown (from left to right in the figure, they correspond to groups B-1, B-2, B-3, B-4, and B-5). In treatment group B-1, the axillary buds in MS medium showed smooth, tender green leaves. In treatment groups B-2, B-3, WPM, and B-5, the axillary buds were small and the leaves were yellowish. In treatment group B-4, the axillary buds in N6 medium germinated and the leaves were yellowish and vitrified. In treatment 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, axillary buds that have sprouted 2-3 fully expanded leaves and grown to 2cm were selected, cut off from the stem segment, and inoculated 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 is 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 adventitious bud proliferation of *Machilus macrantha*. The proliferation coefficient of adventitious buds increases with increasing 6-BA concentration, but high concentrations of 6-BA resulted in numerous tender green adventitious buds, pale green leaves, and a transparent, water-soaked appearance, making them unsuitable for subsequent cultivation. Under higher concentrations of the cytokinin 6-BA and TDZ, the buds of *Machilus macrantha* grew in a ginger-like, sheet-like pattern, exhibiting strong differentiation ability, clear basal callus, and small leaves (C-13 treatment group). Images of adventitious bud clusters from several representative treatment groups during the initial induction phase are shown below. Figure 3 As shown, these correspond to processing 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 control, and seedling cultivation The adventitious buds of *Machilus macrantha* are all clustered (adventitious bud clusters), with dense and short buds and leaves clearly enclosing the stem, which is not conducive to later rooting. Furthermore, with increasing subculturing, more and more metabolic products accumulate around the inserted culture medium, causing the medium to turn black and brown spots to appear on the buds and leaves, severely inhibiting the growth of propagation bottle seedlings. Figure 4 This shows adventitious buds that have turned brown after being subcultured three times 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. From Table 4, Figure 5It can be seen that the addition of adenosine monophosphate (AMP) and activated carbon (AC) to the culture medium has a significant anti-browning effect, and also promotes the high growth of clustered shoots, with stems becoming longer and thicker, and leaves changing from light green to dark green, and from thin to thick and from narrow to wide. 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 other antioxidant treatments was less than 1 cm, the browning rate was higher than 50%, and there were no significant changes in the growth of strong seedlings. Figure 6 (Comparison of D-4 treatment group and 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 hardening-off transplanting Healthy 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] Rooting rate and survival rate are 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] From Table 5, Figure 7 and Figure 8 It can be seen that 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 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 inhibits browning. By increasing the concentration of activated carbon as an adsorbent, the adventitious roots change from brown to white, and the browning at the base of the adventitious buds is alleviated, resulting in whiter roots than those without activated carbon. Considering both root quantity and quality, the optimal culture medium for successful transplanting of *Machilus pungens* tissue culture seedlings is 1 / 2 MS + 0.1 mg / L NAA + 0.1 g / L AC. Finally, the transplant survival rate of *Machilus pungens* tissue culture seedlings obtained from the E-4 treatment group is as follows: Figure 9 As shown.

[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 scope of protection 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 treatment of explants: Select healthy, disease-free semi-lignified stem segments from the current year of a single Phoebe bournei plant as explants, and after pruning and sterilization treatment, they are ready for use; (2) Axillary bud induction culture: The explants obtained by pretreatment in step (1) are cut into small segments of 1-2 buds and inoculated into the induction culture medium for induction culture; (3) Adventitious bud subculture: Axillary buds that have sprouted 2-3 fully expanded leaves and grown to 2 cm in length are cut from the stem segment from the induction medium and inoculated into the adventitious bud subculture medium for adventitious bud subculture. The adventitious bud subculture medium is MS as the base medium, with 0.2 mg / L 6-benzylaminopurine, 0.05 mg / L thidiazuron and 0.5 mg / L indolebutyric acid added. During the adventitious bud subculture, a browning prevention and seedling strengthening treatment was performed. The browning prevention and seedling strengthening treatment was performed by adding 0.1 mg / L adenosine monophosphate and 0.2 g / L activated carbon to the adventitious bud subculture culture medium. (4) Rooting culture, hardening and transplanting: Select healthy single adventitious buds of Phoebe bournei obtained by adventitious bud subculture and inoculate them into rooting culture medium to obtain rooted tissue culture seedlings. Then, harden and transplant the rooted tissue culture seedlings. The rooting culture medium is based on 1 / 2MS medium with 0.1-0.2 mg / L naphthaleneacetic acid and 0.1-0.2 mg / L activated carbon added.

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. A method for rapid in vitro propagation of *Phoebe zhennan* stem segments according to any one of claims 1-3, 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.

5. 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.

6. The method for rapid in vitro propagation of *Machilus chinensis* stem segments 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.

7. 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.

8. The method for rapid in vitro propagation of *Machilus chinensis* stem segments according to claim 7, 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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