A tissue culture and rapid propagation method of litsea cubeba with effectively reduced browning and improved rooting rate

Abstract

This invention discloses a rapid propagation method for Litsea cubeba tissue culture that effectively reduces browning and improves rooting rate. The method selects healthy, young stem segments of Litsea cubeba as explants, sterilizes them, and inoculates them into a primary induction medium to induce initial bud formation. These are then inoculated into a subculture proliferation medium to obtain subculture clusters of buds. Single buds are then cut and inoculated into a rooting medium for dark culture to induce rooting. Finally, the seedlings are hardened off and transplanted to obtain tissue culture seedlings ready for nursery. This invention solves the problems of low subculture proliferation coefficient and severe browning by optimizing the explant sterilization measures, the primary induction medium, the subculture proliferation medium, and the rooting medium formulation, reducing the explant browning rate to below 5.0%. Simultaneously, the subculture yields a large number of clustered buds in a short time with a high proliferation coefficient; the tissue culture seedlings have more than four roots, resulting in a high rooting rate; the transplant survival rate is high, and the seedling quality is good. This reduces the industrial-scale production cost of Litsea cubeba tissue culture seedlings and provides technical support for large-scale seedling production and artificial cultivation of Litsea cubeba.

Description

Technical Field

[0001] This invention belongs to the technical field of plant tissue culture, and specifically relates to a rapid propagation method for Litsea cubeba tissue culture that effectively reduces browning and improves rooting rate. Background Technology

[0002] Litsea cubeba ( Litsea cubeba Litsea cubeba is a small aromatic oilseed tree belonging to the genus Lisea in the family Lauraceae. It is mainly distributed in tropical and subtropical regions of Asia, and in my country, primarily in the Yangtze River basin. It is mainly found in the wild and is a valuable wild aromatic oilseed and medicinal tree species. The fruit, flowers, leaves, and bark of Litsea cubeba contain aromatic oils. The volatile aromatic oil obtained through distillation is called Litsea cubeba oil, a raw material for the pharmaceutical, cosmetic, fragrance, and food and fruit preservation industries. The entire plant can be used medicinally, possessing properties of dispelling wind and cold, warming the kidneys and strengthening the stomach, reducing swelling and relieving pain. The flowers, fruits, roots, and bark of Litsea cubeba are extremely fragrant and can be used as excellent seasonings. Litsea cubeba has good water retention and soil control properties, making it suitable for managing soil erosion in sparse forests. It is a tree species that highly integrates ecological, economic, and social benefits, and can be considered an ideal aromatic oilseed small tree species for transforming low-efficiency forests and establishing economic forests.

[0003] Currently, the artificial cultivation of Litsea cubeba is relatively small-scale, mainly due to limitations in the industrial-scale propagation technology of superior seedlings. Existing seedling propagation techniques mainly include seed propagation, cutting propagation, grafting propagation, and tissue culture. However, Litsea cubeba propagated by seed suffers from significant differences in seed maturity, uneven germination, low germination rates, and severe segregation of traits in offspring, making it difficult to maintain the superior traits of the parent plants. Furthermore, Litsea cubeba is dioecious, and it is difficult to distinguish between male and female plants in the early stages of seedling cultivation, leading to an unreasonable ratio and layout of male and female plants, resulting in seedling waste and making it unsuitable for large-scale afforestation or industrial development.

[0004] In the propagation techniques of cutting and grafting, the wounds on the branches of Litsea cubeba are easily oxidized. The phenolic substances in the damaged cut cells are oxidized into quinones, causing the cut surface of the culture to turn brown or dark brown and undergo browning. Moreover, the propagation process is limited by natural factors, resulting in a low propagation coefficient for cutting and grafting of Litsea cubeba.

[0005] Tissue culture propagation allows for the rapid production of large quantities of seedlings with excellent and uniform traits, unrestricted by factors such as the propagation season, and enables the establishment of nurseries with a reasonable male-to-female ratio. Therefore, tissue culture is more suitable for Litsea cubeba cultivation. However, existing tissue culture patents include: CN102227976B, which discloses a method for rapid asexual propagation of Litsea cubeba using tender shoots as explants to cultivate subculture buds, and then using these tissue-cultured subculture buds as cuttings for in vitro rooting; CN120753194A, which discloses a method for establishing a tissue culture seedling system for the medicinal plant Litsea cubeba using terminal buds as explants; and CN116584381B, which discloses a Litsea cubeba embryogenic callus induction culture medium and induction method, using the embryos within immature fresh Litsea cubeba fruits as material, inoculating them into an induction culture medium for induction culture, obtaining embryogenic callus tissue, and then carrying out proliferation culture. These rapid propagation technologies for Litsea cubeba still have the following problems: (1) During tissue culture of Litsea cubeba, when the explants or cultures are cut and inoculated, phenolic substances in the damaged cut cells are oxidized into quinones, causing the cut surface of the culture to turn brown or dark brown and thus undergo browning. Quinones will gradually diffuse into the culture medium, inhibiting the activity of other enzymes and poisoning the entire tissue.

[0006] (2) HgCl2 and alcohol are used for explant disinfection. HgCl2 will cause damage and necrosis to the explant, induce the production and oxidation of more phenolic substances, and aggravate the browning of the explant; at the same time, there will be mercury residue, causing mercury pollution. (3) The induction rate and proliferation coefficient of Litsea cubeba tissue culture are low, proliferation is difficult, the rooting rate of tissue culture seedlings is not high, the seedling cultivation cycle is long, the seedling quality is poor, the production cost is high, and it is difficult to achieve large-scale production of tissue culture seedlings.

[0007] (4) Callus induction and differentiation of Litsea cubeba tissue culture takes a long time, and the induction rate and proliferation coefficient are relatively low. Summary of the Invention

[0008] The purpose of this invention is to provide a rapid propagation method for Litsea cubeba via tissue culture that effectively reduces browning and increases rooting rate. This overcomes the shortcomings of Litsea cubeba, such as the scarcity of superior varieties, severe genetic differentiation of seedlings obtained from seed propagation, severe browning, low proliferation coefficient, low rooting rate, high production cost, and inconsistent seedling quality in tissue culture propagation. This provides technical support for the large-scale production of superior Litsea cubeba through rapid propagation.

[0009] To achieve the above objectives, this invention provides a rapid propagation method for Litsea cubeba tissue culture that effectively reduces browning and improves rooting rate, comprising the following steps: (1) Explant collection and disinfection: On a sunny day, collect semi-lignified branches of healthy female and male plants that are free from diseases and pests. Cut the stem segments with axillary buds into 4cm to 6cm lengths as explants; rinse the surface mud and sand with clean water, and then disinfect them. (2) Primary induction culture: Cut off 1 mm from both ends of the explant obtained in step (1) with scissors and trim it into stem segments with 1 to 2 buds. Insert the segments vertically into the prepared primary induction culture medium and culture one plant per bottle to obtain initial buds. (3) Subculture: When the initial shoot height is 3cm to 6cm, cut off the old leaves, retain the terminal bud and part of the petiole, and prune it into a single shoot with a height of 1.0cm to 2.0cm. Transfer it into the prepared subculture culture medium, and inoculate 5 to 8 plants per bottle. After 20 to 25 days of culture, the subculture bud clusters will be formed. When carrying out subculture culture again, cut off the callus tissue at the bottom of the bud clusters and cut it into single shoots or small bud clusters with 2 to 3 buds. Inoculate them into the subculture culture medium, and inoculate 5 to 8 plants per bottle. (4) Rooting culture: Select healthy terminal buds with a height of ≥1 cm from the subculture buds in step (3), and vertically insert them into the prepared rooting culture medium under sterile conditions. Insert 10 to 15 buds into each bottle and culture for 15 to 20 days to obtain healthy tissue culture rooted seedlings. (5) Hardening and transplanting: When the root length of the tissue culture rooted seedlings in step (4) is ≥1.0 cm and the number of roots is more than 3, the healthy tissue culture rooted seedlings are moved to an outdoor seedling greenhouse for hardening for 5 to 10 days. After that, the root culture medium is washed off with clean water and transplanted into a sterilized substrate seedling container and seedling management is carried out.

[0010] Further, the specific steps of the disinfection treatment described in step (1) are as follows: After cleaning the mud and sand off the surface of the explant, first soak it in a 2-3% volume concentration of detergent for 10-15 minutes, then soak it in a 2-5% volume concentration of benzalkonium chloride solution for 30-60 minutes, rinse it with running water for 1-2 hours, rinse it with sterile water 3-5 times, then seal it in a sterile culture dish and refrigerate it at 7-9℃ for 10-15 hours, then soak it in a 70-75% volume concentration of alcohol solution for 10-30 seconds on a clean bench, rinse it with sterile water 3-5 times, and finally disinfect it in a 4-6% volume concentration of sodium hypochlorite solution for 25-30 minutes, rinse it with sterile water 3-5 times, place the explant material on sterilized filter paper to absorb the moisture, and set it aside.

[0011] Furthermore, the control conditions for the primary induction culture described in step (2) are as follows: dark culture for 5 to 7 days, temperature of 25±3℃, followed by light culture for 8 to 10 days, light intensity of 1600 to 2500 Lx, and light duration of 12 to 14 h / d.

[0012] Further, the stem segment length in step (2) is 1cm to 1.5cm, and the formula of the primary induction medium is: MS, 0.5 to 2.5 mg / L TDZ (thidiazuron), 0.1 to 0.5 mg / L NAA, 0.5 to 2.0 mg / L KT (kinetin), 0.005 to 0.02 mg / L ascorbic acid, 0.5 to 2 g / L PVP (polyvinylpyrrolidone), 3 to 5 g / L agar, 28 to 32 g / L sugar, and pH 5.8 to 6.2.

[0013] Further, the formulation of the subculture proliferation medium in step (3) is as follows: MS, 0.5-2.0 mg / L 6-BA, 0.3-0.5 mg / L NAA, 0.5-3.0 mg / L KT, 3-5 g / L agar, 28-32 g / L sugar, 0.01-0.02 mg / L riboflavin, 0.005-0.02 mg / L ascorbic acid, 0.5-2 g / L Flower Treasure No. 1, and pH 5.8-6.2.

[0014] Furthermore, in step (3), the controlled conditions for cultivation are as follows: temperature 25±3℃, light intensity 2500~9000 Lx, and light duration 12~16 h / d.

[0015] Further, the rooting medium in step (4) is formulated as follows: 1 / 2 WPM, 1-1.5 mg / L IBA (indole-3-butyric acid), 0.5-1.0 mg / L IAA (indole-3-acetic acid), 1-2.0 g / L activated carbon, 0.5-2 g / L PVP, 3-5 g / L agar, 14-16 g / L sugar, and pH 5.8-6.2.

[0016] Furthermore, in step (4), the culture control conditions are as follows: dark culture for 5 days, followed by light culture for 10 to 15 days, with a temperature of 25±3℃, a light intensity of 2500 to 9000 Lx, and a light duration of 12 to 16 h / d.

[0017] Furthermore, 3 days before transplanting the seedlings described in step (5), the seedling substrate is thoroughly disinfected by rinsing with a potassium permanganate solution with a volume concentration of 0.5-1.0%, and then rinsed with clean water to remove the potassium permanganate solution 1 day later.

[0018] Furthermore, in step (5), the seedling substrate is composed of yellow mud, coconut coir, and peat soil mixed in a volume ratio of 1:1 to 2:3.

[0019] Further, in step (5), the seedling management method is as follows: place the seedlings in a nursery with a shade net, and spray water once or twice a day, morning and evening; after 7 days, spray with a 0.3% to 0.5% compound fertilizer (15-15-15) aqueous solution, spraying once or twice every 10 days; within one week after transplanting, maintain the light transmittance at 40% to 50%, and gradually increase the light after one week. One month after transplanting, the light transmittance increases to 80% to 90%.

[0020] Furthermore, in step (5), the seedling container is a non-woven bag with a diameter of 7cm to 9cm and a height of 9cm to 12cm.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The explant disinfection method, hormone type and concentration, low temperature treatment, dark culture, culture conditions, and addition of an appropriate amount of anti-browning agent used in this invention can effectively reduce the browning rate of Litsea cubeba, improve the explant induction rate, subculture bud proliferation coefficient, rooting rate and rooting quality of Litsea cubeba, and can cultivate a large number of Litsea cubeba seedlings with consistent traits in a short time, realize large-scale production, meet production needs, and improve economic benefits.

[0022] 2. This invention uses superior single plants and robust current-year stem segments of Litsea cubeba as the source of explants. The explants are sterilized with benzalkonium chloride and NaClO solution to prevent Hg2+ from damaging the explant tissue or aggravating cell necrosis on the cut surface, and to avoid inducing the production and oxidation of more phenolic substances, thereby reducing browning of the explants.

[0023] 3. The selection of the components and ratios of the initial culture medium in this invention promotes the growth of induced shoot clusters. PVP and ascorbic acid can adsorb, complex, flocculate, and encapsulate phenolic substances, which can effectively reduce and prevent the phenols produced during the tissue culture of Litsea cubeba from being oxidized into quinones upon contact with oxygen, thereby reducing the browning rate. The browning rate is controlled below 5%, and in the best case, it can reach below 1%.

[0024] 4. The composition and ratio of the subculture medium and the culture conditions of this invention enable the induced bud proliferation to produce more and faster clustered buds, effectively increasing the proliferation coefficient. The added Flower Treasure No. 1 promotes strong stems and the growth of stems and leaves, thereby shortening the culture time and saving production costs. Healthy subculture buds are inoculated into a rooting medium containing IBA, IAA, activated carbon, and PVP for dark culture, which is beneficial for obtaining a large number of robust and well-developed rooted tissue culture seedlings of Litsea cubeba.

[0025] 5. The in vitro regeneration process of the present invention does not require inducing callus tissue to redifferentiate into buds. Instead, it directly uses the method of propagating buds to multiply and then root and grow seedlings, saving time and operation steps and making the operation simpler.

[0026] 6. The culture medium formula obtained by this invention and the specific light and temperature conditions can adapt to the growth characteristics of Litsea cubeba tissue culture seedlings, promote seedling growth, and the initial bud induction rate can reach 98%. After 20 to 25 days of subculture and proliferation culture, cluster buds can be formed with a proliferation coefficient as high as 8.9. After a single bud is inoculated into the rooting medium, the rooting rate is as high as 98% after 20 days, with more than 5 roots, and the roots are thick and strong. The transplant survival rate can reach 98.3%, which greatly shortens the seedling cultivation cycle and can obtain a large number of Litsea cubeba tissue culture seedlings in a short period of time. Attached Figure Description

[0027] Figure 1 Example 1: Induction of buds from tissue culture stem segments of Litsea cubeba.

[0028] Figure 2 Example 1: Litsea cubeba seedlings propagated by subculture.

[0029] Figure 3 Example 1: Litsea cubeba tissue culture rooting bottle seedlings.

[0030] Figure 4 Example 1: Litsea cubeba tissue culture transplanted seedlings.

[0031] Figure 5 This is a diagram of container seedlings successfully transplanted from tissue culture of Litsea cubeba in Example 1.

[0032] Figure 6 Example 1: Large seedlings of Litsea cubeba transplanted from tissue culture.

[0033] Figure 7 As a comparative example, buds were induced from tissue-cultured stem segments of Litsea cubeba.

[0034] Figure 8 As a comparative example, buds were induced from tissue-cultured stem segments of Litsea cubeba.

[0035] Figure 9 To compare the results, buds were induced from tissue-cultured stem segments of Litsea cubeba in Example 3.

[0036] Figure 10 As a comparative example, buds were induced from tissue-cultured stem segments of Litsea cubeba.

[0037] Figure 11 To compare the results, buds were induced from tissue-cultured stem segments of Litsea cubeba in Example 5.

[0038] Figure 12 Example 2: Litsea cubeba seedlings propagated by subculture.

[0039] Figure 13Example 3: Litsea cubeba seedlings were propagated and cultured in bottles.

[0040] Figure 14 Example 4: Litsea cubeba seedlings propagated by subculture.

[0041] Figure 15 As a comparative example, Litsea cubeba seedlings were cultured in subculture bottles for propagation.

[0042] Figure 16 Example 2: Litsea cubeba seedlings were propagated and cultured in bottles.

[0043] Figure 17 For comparison, Litsea cubeba seedlings were propagated by subculture in bottles.

[0044] Figure 18 For comparison, Litsea cubeba seedlings were cultured in subculture bottles.

[0045] Figure 19 Five Litsea cubeba seedlings were cultured in subculture bottles for comparison.

[0046] Figure 20 Example 2: Litsea cubeba tissue culture rooting bottle seedlings.

[0047] Figure 21 Example 3: Litsea cubeba tissue culture rooting bottle seedlings.

[0048] Figure 22 Example 4: Litsea cubeba tissue culture rooting bottle seedlings.

[0049] Figure 23 As a comparative example, Litsea cubeba tissue culture rooting bottle seedlings.

[0050] Figure 24 Example 2: Litsea cubeba tissue culture rooting bottle seedlings.

[0051] Figure 25 Example 3: Litsea cubeba tissue culture rooting bottle seedlings.

[0052] Figure 26 For comparison, there are four Litsea cubeba tissue culture rooting bottle seedlings.

[0053] Figure 27 Five Litsea cubeba tissue culture rooting bottle seedlings were used as a control. Detailed Implementation

[0054] The following embodiments are intended to provide a more comprehensive understanding of the invention by those skilled in the art, but do not limit the invention in any way. However, those skilled in the art should understand that various changes in form and detail can be made without departing from the scope defined by the claims.

[0055] Example 1: A rapid propagation method for Litsea cubeba tissue culture that effectively reduces browning and improves rooting rate includes the following steps: (1) Explant collection and disinfection: On a sunny day, healthy, disease-free, semi-lignified branches of female and male plants were collected. The stem segments with axillary buds were cut into 4cm lengths as explants. After cleaning the mud and sand off the surface of the explants, they were first soaked in 2% detergent solution for 10 min, then soaked in 2% benzalkonium chloride solution for 30 min, rinsed with running water for 1 h, rinsed with sterile water 5 times, then sealed in a sterile culture dish and refrigerated at 7°C for 10 h. Then, on a clean bench, they were soaked in 75% alcohol solution for 10 s, rinsed with sterile water 5 times, and finally disinfected in 4% sodium hypochlorite solution for 25 min, rinsed with sterile water 5 times. The explant material was then placed on sterile filter paper to absorb the moisture. (2) Primary induction culture: After sterilization, the stem segments were cut off at both ends of the buds and the wound surface, and the segments were trimmed into stem segments with 1-2 nodes or axillary buds. The segments were then inoculated onto the primary induction culture medium by oblique insertion, one plant per bottle. The primary induction culture medium formula was: MS + TDZ 1.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + PVP 1.0 g / L + ascorbic acid 0.01 mg / L + agar 3.7 g / L + sugar 30 g / L, pH = 5.8 - 6.2. The plants were first cultured in the dark for 5 days at 23℃, and then cultured under light for 8 days at a light intensity of 1500 LX and a light duration of 10 h / d to obtain the initial buds.

[0056] (3) Subculture: When the initial shoot height is 3-6 cm, cut off the old leaves, retain the terminal bud and part of the petiole, and prune into single shoots with a height of 1-2 cm. Transfer to the prepared subculture proliferation medium, 5 plants per bottle. The formula of the subculture proliferation medium is: MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L + KT 1.5 mg / L + agar 3.7 g / L + sugar 28 g / L + riboflavin 0.01 mg / L + ascorbic acid 0.01 mg / L + Flower Treasure No. 1 0.1 g / L, pH = 5.8-6.2. After 20 days of culture, the proliferation forms cluster shoots with a proliferation coefficient of 8.9. When carrying out subculture proliferation culture again, cut off the callus tissue at the bottom of the cluster shoots, cut them into single shoots or small cluster shoots with 2-3 buds, and inoculate them into the subculture proliferation medium, 5 plants / cluster per bottle. The culture control conditions are: temperature 23℃, light intensity 4000 Lx, illumination time 16 h / d.

[0057] (4) Rooting culture: Select healthy single shoots with a shoot height ≥1 cm from the subculture bud clusters and vertically insert them into the prepared rooting culture medium under sterile conditions for rooting culture. 10 shoots are inserted into each bottle. The formula of the rooting culture medium is: 1 / 2 WPM + IBA 1.0 mg / L + IAA 1.0 mg / L + PVP 1.0 g / L + activated carbon 1.0 g / L + agar 3.7 g / L + sugar 15 g / L, pH = 5.8-6.2. Culture in the dark for 5 days under the environmental conditions of 23℃, light intensity 300 Lx, and light time 14 h / d. Then continue to culture under light. The culture control conditions are: temperature 23℃, light intensity 2500 Lx, and light time 14 h / d. Roots will grow after 8 days.

[0058] (5) Hardening off and transplanting: When the root length of the tissue culture rooted seedlings is ≥1 cm and the number of roots is more than 3, the healthy tissue culture rooted seedlings are transplanted to an outdoor seedling greenhouse for hardening off for 6 days. After that, the culture medium on the roots is washed off with clean water and transplanted into sterilized substrate seedling containers. The seedling substrate is composed of yellow clay, coconut coir and peat moss in a volume ratio of 1:1:2. It is filled into non-woven bags with a diameter of 7-9 cm and a height of 9-12 cm. Three days before transplanting, the seedling substrate is thoroughly disinfected by watering with a 0.5% potassium permanganate solution. One day later, the potassium permanganate solution is washed off with clean water. After transplanting, place the seedlings in a nursery with shade netting and spray them with water 1-2 times each morning and evening. Within one week after transplanting, maintain the light transmittance at 40%. After one week, gradually increase the light exposure. One month after transplanting, increase the light transmittance to 80%. Seven days later, spray with a 0.3% solution of compound fertilizer (15-15-15) every 10 days, 1-2 times.

[0059] Example 2: A rapid propagation method for Litsea cubeba tissue culture that effectively reduces browning and improves rooting rate includes the following steps: (1) Explant collection and disinfection: On a sunny day, healthy, disease-free, semi-lignified branches of female and male plants were collected. The stem segments with axillary buds were cut into 4.5 cm lengths as explants. After cleaning the mud and sand off the surface of the explants, they were first soaked in 2.5% detergent solution for 12 min, then soaked in 3% benzalkonium chloride solution for 35 min, rinsed with running water for 1.2 h, rinsed 3 times with sterile water, then sealed in a sterile culture dish and refrigerated at 7°C for 11 h. Then, on a clean bench, they were soaked in 70% alcohol solution for 15 s, rinsed 3 times with sterile water, and finally disinfected in 4.5% sodium hypochlorite solution for 27 min, rinsed 3 times with sterile water, and the explant material was placed on sterile filter paper to absorb the moisture. (2) Primary induction culture: After sterilization, the stem segments were cut off at both ends of the buds and the wound surface, and the stem segments with 1-2 nodes or axillary buds were cut. The segments were inoculated on the primary induction culture medium by oblique insertion, one plant per bottle. The formula of the primary induction culture medium was: MS + TDZ 1.0 mg / L + NAA 0.1 mg / L + KT 1.5 mg / L + PVP 1.0 g / L + ascorbic acid 0.005 mg / L + agar 4.0 g / L + sugar 30 g / L, pH = 5.8 - 6.2. The plants were first cultured in the dark for 6 days at 24℃, and then cultured under light for 10 days at a light intensity of 1800 LX and a light duration of 10 h / d to obtain the initial buds.

[0060] (3) Subculture: When the initial shoot height is 3-6 cm, cut off the old leaves, retain the terminal bud and part of the petiole, and prune into single shoots with a height of 1-2 cm. Transfer them to the prepared subculture medium, 5 plants per bottle. The formula of the subculture medium is: MS + 6-BA 0.5 mg / L + NAA 0.3 mg / L + KT 2.0 mg / L + agar 4.0 g / L + sugar 30 g / L + riboflavin 0.02 mg / L + ascorbic acid 0.005 mg / L + Flower Treasure No. 1 0.8 At a concentration of g / L and pH = 5.8-6.2, after 20 days of culture, the buds proliferated and formed clusters with a proliferation coefficient of 8.5. When carrying out subculture again, the callus tissue at the base of the clusters was removed, and the buds were divided into single buds or small clusters with 2-3 buds. These clusters were then inoculated into the subculture medium, with 5 plants / clump per bottle. The culture conditions were controlled as follows: temperature 24℃, light intensity 5000 Lx, and light duration 15 h / d.

[0061] (4) Rooting culture: Select healthy single shoots with a shoot height ≥1 cm from the subculture bud clusters and vertically insert them into the prepared rooting culture medium under sterile conditions for rooting culture. 10 shoots are inserted into each bottle. The formula of the rooting culture medium is: 1 / 2 WPM + IBA 0.5 mg / L + IAA 0.5 mg / L + PVP 1.0 g / L + activated carbon 1.5 g / L + agar 4.0 g / L + sugar 15 g / L, pH = 5.8-6.2. Culture in the dark for 5 days under the environmental conditions of 24℃, light intensity 400 Lx, and light time 13 h / d. Then continue to culture under light. The culture control conditions are: temperature 24℃, light intensity 3000 Lx, and light time 13 h / d. Roots will grow after 10 days.

[0062] (5) Hardening off and transplanting: When the root length of the tissue culture rooted seedlings is ≥1 cm and the number of roots is more than 3, the healthy tissue culture rooted seedlings are transplanted to an outdoor seedling greenhouse for hardening off for 7 days. After that, the culture medium on the roots is washed off with clean water and transplanted into sterilized substrate seedling containers. The seedling substrate is composed of yellow clay, coconut coir and peat moss in a volume ratio of 1:1:2. It is filled into non-woven bags with a diameter of 7-9 cm and a height of 9-12 cm. Three days before transplanting, the seedling substrate is thoroughly disinfected by watering with a 0.6% potassium permanganate solution. One day later, the potassium permanganate solution is washed off with clean water. After transplanting, place the seedlings in a nursery with shade netting and spray them with water 1-2 times each morning and evening. Within one week after transplanting, maintain the light transmittance at 40%. After one week, gradually increase the light exposure. One month after transplanting, increase the light transmittance to 80%. Seven days later, spray with a 0.3% solution of compound fertilizer (15-15-15) every 10 days, 1-2 times.

[0063] Example 3: A rapid propagation method for Litsea cubeba tissue culture that effectively reduces browning and improves rooting rate includes the following steps: (1) Explant collection and disinfection: On a sunny day, healthy, disease-free, semi-lignified branches of female and male plants were collected. The stem segments with axillary buds were cut into 5.0 cm lengths as explants. After cleaning the mud and sand off the surface of the explants, they were first soaked in 3% detergent solution for 10 min, then soaked in 3.5% benzalkonium chloride solution for 40 min, rinsed with running water for 1.5 h, rinsed with sterile water 5 times, then sealed in a sterile culture dish and refrigerated at 8℃ for 13 h. Then, on a clean bench, they were soaked in 75% alcohol solution for 15 s, rinsed with sterile water 3 times, and finally disinfected in 5% sodium hypochlorite solution for 28 min, rinsed with sterile water 4 times. The explant material was then placed on sterile filter paper to absorb the moisture. (2) Primary induction culture: After sterilization, the stem segments were cut off at both ends of the buds and the wound surface, and the stem segments with 1-2 nodes or axillary buds were cut off. The segments were then inoculated onto the primary induction culture medium by oblique insertion, one plant per bottle. The formula of the primary induction culture medium was: MS + TDZ 0.5 mg / L + NAA 0.1 mg / L + KT 1.0 mg / L + PVP 1.5 g / L + ascorbic acid 0.015 mg / L + agar 4.2 g / L + sugar 28 g / L, pH = 5.8 - 6.2. The explants were first cultured in the dark for 7 days at 25℃, and then cultured under light for 10 days at a light intensity of 2000 LX and a light duration of 12 h / d to obtain the initial buds.

[0064] (3) Subculture: When the initial shoot height is 3-6 cm, cut off the old leaves, retaining the terminal bud and part of the petiole, and prune into single shoots with a height of 1-2 cm. Transfer to the prepared subculture medium, 5 plants per bottle. The formula of the subculture medium is: MS + 6-BA 1.5 mg / L + NAA 0.3 mg / L + KT 0.5 mg / L + agar 4.2 g / L + sugar 30 g / L + ascorbic acid 0.015 mg / L + 0.015 mg / L riboflavin + Flower Treasure No. 1 1.0 At a concentration of g / L and pH = 5.8-6.2, after 20 days of culture, the buds proliferated and formed clusters with a proliferation coefficient of 8.0. When carrying out subculture again, the callus tissue at the base of the clusters was removed, and the buds were divided into single buds or small clusters with 2-3 buds. These clusters were then inoculated into the subculture medium, with 5 plants / clump per bottle. The culture conditions were controlled as follows: temperature 25℃, light intensity 6000 Lx, and light duration 14 h / d.

[0065] (4) Rooting culture: Select healthy single shoots with a shoot height ≥1 cm from the subculture bud clusters and vertically insert them into the prepared rooting culture medium under sterile conditions for rooting culture. 10 shoots are inserted into each bottle. The formula of the rooting culture medium is: 1 / 2 WPM + IBA 1.5 mg / L + IAA 1.0 mg / L + PVP 1.5 g / L + activated carbon 1.0 g / L + agar 4.2 g / L + sugar 15 g / L, pH = 5.8-6.2. Culture in the dark for 6 days under the environmental conditions of 25℃, light intensity 500 Lx, and light time 14 h / d. Then continue to culture under light. The culture control conditions are: temperature 25℃, light intensity 3500 Lx, and light time 13 h / d. Roots will grow after 11 days.

[0066] (5) Hardening off and transplanting: When the root length of the tissue culture rooted seedlings is ≥1 cm and the number of roots is more than 3, the healthy tissue culture rooted seedlings are transplanted to an outdoor seedling greenhouse for hardening off for 8 days. After that, the culture medium on the roots is washed off with clean water and transplanted into sterilized substrate seedling containers. The seedling substrate is composed of yellow clay, coconut coir and peat moss in a volume ratio of 1:1:2. It is filled into a non-woven bag with a diameter of 7-9 cm and a height of 9-12 cm. Three days before transplanting, the seedling substrate is thoroughly disinfected by watering with a 0.8% potassium permanganate solution. One day later, the potassium permanganate solution is washed off with clean water. After transplanting, place the seedlings in a nursery with shade netting and spray them with water 1-2 times each morning and evening. Within one week after transplanting, maintain the light transmittance at 40%. After one week, gradually increase the light exposure. One month after transplanting, increase the light transmittance to 80%. Seven days later, spray with a 0.3% solution of compound fertilizer (15-15-15) every 10 days, 1-2 times. Example 4 A rapid propagation method for Litsea cubeba tissue culture that effectively reduces browning and improves rooting rate includes the following steps: (1) Explant collection and disinfection: On a sunny day, healthy, disease-free, semi-lignified branches of female and male plants were collected. The stem segments with axillary buds were cut into 6cm lengths as explants. After cleaning the mud and sand off the surface of the explants, they were first soaked in 2.5% detergent solution for 13 min, then soaked in 4% benzalkonium chloride solution for 45 min, rinsed with running water for 1.8 h, rinsed with sterile water 4 times, then sealed in a sterile culture dish and refrigerated at 9℃ for 13 h. Then, on a clean bench, they were soaked in 70% alcohol solution for 25 s, rinsed with sterile water 5 times, and finally disinfected in 5.5% sodium hypochlorite solution for 28 min, rinsed with sterile water 5 times. The explant material was then placed on sterile filter paper to absorb the moisture. (2) Primary induction culture: After sterilization, the stem segments were cut off at both ends of the buds and the wound surface, and the stem segments with 1-2 nodes or axillary buds were cut. The segments were inoculated on the primary induction culture medium by oblique insertion, one plant per bottle. The formula of the primary induction culture medium was: MS + TDZ 2.0 mg / L + NAA 0.5 mg / L + KT 0.5 mg / L + PVP 2.0 g / L + ascorbic acid 0.02 mg / L + agar 4.5 g / L + sugar 29 g / L, pH = 5.8-6.2. The plants were first cultured in the dark for 6 days at a temperature of 26℃, and then cultured under light for 9 days at a light intensity of 2200 LX and a light duration of 11 h / d to obtain the initial buds.

[0067] (3) Subculture: When the initial shoot height is 3-6 cm, cut off the old leaves, retain the terminal bud and part of the petiole, and prune into single shoots with a height of 1-2 cm. Transfer to the prepared subculture proliferation medium, 5 plants per bottle. The formula of the subculture proliferation medium is: MS + 6-BA 2.0 mg / L + NAA 0.5 mg / L + KT 2.5 mg / L + agar 4.5 g / L + sugar 32 g / L + ascorbic acid 0.02 mg / L + riboflavin 0.02 mg / L + Flower Treasure No. 1 1.5 g / L, pH = 5.8-6.2. After 20 days of culture, the bud clusters will be formed with a proliferation coefficient of 7.5. When carrying out subculture proliferation again, cut off the callus tissue at the bottom of the bud clusters and cut them into single shoots or small bud clusters with 2-3 buds. Inoculate them into the subculture proliferation medium, 5 plants / clump per bottle. The culture control conditions are: temperature 26℃, light intensity 7000 Lx, illumination time 13 h / d.

[0068] (4) Rooting culture: Select robust single shoots with a height ≥1 cm from the subculture bud clusters, and vertically insert them into the prepared rooting medium under aseptic conditions for rooting culture. 10 shoots are inserted per bottle. The rooting medium formula is: 1 / 2 WPM + IBA 1.5 mg / L + IAA 0.5 mg / L + PVP 1.0 g / L + activated carbon 1.5 g / L + agar 4.5 g / L + sugar 15 g / L, pH 15. =5.8-6.2, cultured in the dark for 7 days under the following conditions: temperature 26℃, light intensity 600 Lx, light duration 13 h / d. Then, cultured under light for another 13 days under the following conditions: temperature 26℃, light intensity 5000 Lx, light duration 14 h / d. Roots will grow after 13 days.

[0069] (5) Hardening off and transplanting: When the root length of the tissue culture rooted seedlings is ≥1 cm and the number of roots is more than 3, the healthy tissue culture rooted seedlings are transplanted to an outdoor seedling greenhouse for hardening off for 9 days. After that, the culture medium on the roots is washed off with clean water and transplanted into sterilized substrate seedling containers. The seedling substrate is composed of yellow clay, coconut coir and peat moss in a volume ratio of 1:1:2. It is filled into non-woven bags with a diameter of 7-9 cm and a height of 9-12 cm. Three days before transplanting, the seedling substrate is thoroughly disinfected by rinsing with a 0.9% potassium permanganate solution. One day later, the potassium permanganate solution is rinsed off with clean water. After transplanting, place the seedlings in a nursery with shade netting and spray them with water 1-2 times each morning and evening. Within one week after transplanting, maintain the light transmittance at 40%. After one week, gradually increase the light exposure. One month after transplanting, increase the light transmittance to 80%. Seven days later, spray with a 0.3% solution of compound fertilizer (15-15-15) every 10 days, 1-2 times. Comparative Example 1: The method is basically the same as in Example 1, except that the subculture, rooting culture method and transplanting substrate are based on the formula disclosed in "The method for rapid asexual propagation of Litsea cubeba disclosed in CN102227976B", and the transplanting survival rate is 70.7%.

[0070] Comparative Example 2: The method was basically the same as in Example 1, except that the induction, subculture proliferation, rooting culture medium and transplanting substrate were based on the formula disclosed in "A Method for Establishing a Tissue Culture Seedling System of Litsea cubeba, a Medicinal Plant" disclosed in CN120753194A, and the transplanting survival rate was 78.3%.

[0071] Comparative Example 3: Steps (1) and (5) are the same as in Example 1; the difference is that... (2) Primary induction culture: After sterilization, the stem segments were cut off at both ends of the buds and the wound surface, and the stem segments with 1-2 nodes or axillary buds were cut. The segments were inoculated on the primary induction culture medium by oblique insertion, one plant per bottle. The formula of the primary induction culture medium was: MS + TDZ 1.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + agar 3.7 g / L + sugar 30 g / L, pH = 5.8-6.2. The plants were first cultured in the dark for 5 days at 23℃, and then cultured under light for 8 days at a light intensity of 1500 LX and a light duration of 10 h / d to obtain the initial buds.

[0072] (3) Subculture: When the initial shoot height is 3-6 cm, cut off the old leaves, retain the terminal bud and part of the petiole, and prune into single shoots with a height of 1-2 cm. Transfer them into the prepared subculture culture medium, 5 plants per bottle. The formula of the subculture culture medium is: MS + 6-BA 1.5 mg / L + NAA 0.3 mg / L + KT 0.5 mg / L + agar 4.2 g / L + sugar 30 g / L + Flower Treasure No. 1 1.0 g / L, pH = 5.8-6.2. After 20 days of culture, the shoots will proliferate and form clusters. When carrying out subculture again, cut off the callus tissue at the bottom of the clusters and cut them into single shoots or small clusters with 2-3 shoots. Inoculate them into the subculture culture medium, 5 plants / cluster per bottle. The culture control conditions are: temperature 23℃, light intensity 4000 Lx, and light duration 16 h / d.

[0073] (4) Rooting culture: Select healthy single shoots with a shoot height ≥1 cm from the subculture bud clusters and insert them vertically into the prepared rooting culture medium under sterile conditions for rooting culture. 10 shoots are inserted into each bottle. The formula of the rooting culture medium is: 1 / 2 WPM + IBA 1.0 mg / L + IAA 1.0 mg / L + agar 3.7 g / L + sugar 15 g / L, pH = 5.8-6.2. Incubate in the dark for 5 days at a temperature of 23℃, a light intensity of 300 Lx, and a light duration of 14 h / d. Then continue to culture under light at a light intensity of 2500 Lx and a light duration of 14 h / d. Roots will grow after 10 days.

[0074] Comparative Example 4: Steps (1) and (5) are the same as in Example 1; the difference is that... (2) Primary induction culture: After sterilization, the stem segments were cut off at both ends of the buds and the wound surface, and the stem segments with 1-2 nodes or axillary buds were cut off. The segments were inoculated on the primary induction culture medium by oblique insertion, one plant per bottle. The formula of the primary induction culture medium was: WPM + TDZ 1.5 mg / L + NAA 0.3 mg / L + KT 1.0 mg / L + PVP 0.5 g / L + agar 3.7 g / L + sugar 30 g / L, pH = 5.8-6.2. The plants were first cultured in the dark for 5 days at 23℃, and then cultured under light for 10 days at a light intensity of 1500 LX and a light duration of 10 h / d to obtain the initial buds.

[0075] (3) Subculture: When the initial shoot height is 3-6 cm, cut off the old leaves, retain the terminal bud and part of the petiole, and prune into single shoots with a height of 1-2 cm. Transfer them into the prepared subculture culture medium, 5 plants per bottle. The formula of the subculture culture medium is: WPM + 6-BA 1.5 mg / L + NAA 0.3 mg / L + KT 0.5 mg / L + agar 4.2 g / L + sugar 30 g / L + Flower Treasure No. 1 1.0 g / L, pH = 5.8-6.2. After 20 days of culture, the shoots will proliferate and form clusters. When carrying out subculture again, cut off the callus tissue at the bottom of the clusters and cut them into single shoots or small clusters with 2-3 shoots. Inoculate them into the subculture culture medium, 5 plants / cluster per bottle. The culture control conditions are: temperature 23℃, light intensity 4000 Lx, and light duration 16 h / d.

[0076] (4) Rooting culture: Select healthy single shoots with a shoot height ≥1 cm from the subculture bud clusters and vertically insert them into the prepared rooting culture medium under sterile conditions for rooting culture. 10 shoots are inserted into each bottle. The formula of the rooting culture medium is: 1 / 2 WPM + IBA 1.0 mg / L + IAA 1.0 mg / L + activated carbon 1.0 g / L + agar 3.7 g / L + sugar 15 g / L, pH = 5.8-6.2. Incubate in the dark for 5 days at a temperature of 23℃, a light intensity of 300 Lx, and a light duration of 14 h / d. Then continue to culture under light with a light intensity of 2500 Lx and a light duration of 14 h / d. Roots will grow after 18 days.

[0077] Comparative Example 5: Steps (2), (3), and (5) are the same as in Example 1; the difference is that... (1) Explant disinfection: The explant pretreatment was the same as in Example 1, except that it was disinfected with 75% alcohol for 20 seconds, rinsed with sterile water 5 times, and then disinfected with 0.1% HgCl2 for 10 minutes. (4) Rooting culture: Select healthy single shoots with a shoot height ≥1 cm from the subculture bud clusters, and vertically insert them into the prepared rooting culture medium under sterile conditions for rooting culture. 10 shoots are inserted into each bottle. The formula of the rooting culture medium is: 1 / 2 WPM + IBA 1.0 mg / L + IAA 1.0 mg / L + PVP 2.0 g / L + activated carbon 1.0 g / L + agar 3.7 g / L + sugar 15 g / L, pH = 5.8-6.2. No dark culture is performed. The temperature is 23℃, the light intensity is 2500 Lx, and the light time is 14 h / d. Roots will grow after 16 days. In Examples 1-4 and Comparative Examples 1-5, 100 bottles of tissue culture seedlings were inoculated for primary culture. The browning of the explants was observed and the browning rate was calculated. After the primary culture, the quinone content in the culture medium was measured, and the results are shown in Tables 1 and 2. For the subcultured seedlings, 100 bottles were randomly selected from each group for subculture to observe the number of shoot clusters, and the results are shown in Table 3. For the rooted seedlings, 10 bottles were randomly selected from each group, and the number of roots (root length ≥ 1 cm) and the rooting rate were counted, as shown in Tables 1 and 2. After hardening off, 1000 seedlings were transplanted from each experimental group, and the survival rate was calculated after 30 days.

[0078] The calculation formula is as follows: Browning rate (%) = (Total browned explants / Total number of explants inoculated) × 100% Initial bud induction rate / % = (Number of explants induced to produce initial buds / Total number of inoculated explants) × 100%; Proliferation coefficient = number of buds that grow to a height of 0.5cm-1cm that can be cut / total number of single buds at the time of inoculation; Rooting rate / % = (Number of rooted buds / Total number of inoculated buds) × 100%; Table 1. Quinone content, browning rate, induction rate, rooting status, and transplanting rate in Examples 1-5

[0079] The browning of explants is mainly caused by polyphenol oxidase acting on natural phenolic substrates to form quinones. Anti-browning agents can inhibit polyphenol oxidase to a certain extent in preventing browning of explants, thus preventing the phenols from being oxidized.

[0080] As shown in Table 1, Example 1 exhibits the best results, and the growth at each stage is as follows: Figures 1-6As shown, the average content of quinones in Examples 1 to 4 gradually increased, and the browning rate also gradually increased. Example 1 had the lowest average content of quinones and the lowest browning rate of explants, at 1%. This indicates that appropriate low-temperature treatment and the addition of appropriate amounts of anti-browning agents: PVP (1.0 g / L), ascorbic acid (0.01 mg / L), and activated carbon (1.0 g / L) can effectively reduce the browning rate. If too little anti-browning agent is added (Example 2) or too much (Examples 3-5), the average content of quinones is high, the browning of explants is more severe, the effect of preventing browning is poor, and thus it is detrimental to the growth of explants, resulting in a decrease in induction rate, rooting rate, and transplant survival rate.

[0081] The rooting rate effect diagrams of Examples 2-4 are as follows: Figure 20 , Figure 21 , Figure 22 As shown.

[0082] Table 2. Average quinone content, browning rate, induction rate, rooting status, and transplanting rate of Examples 1 and Comparative Examples 1-5

[0083] As shown in Table 2, compared with Comparative Examples 1-5, Example 1 demonstrates that the method of the present invention resulted in the lowest average content of quinones in the primary induction medium and the lowest browning rate. Compared with Example 1, Comparative Example 5 used 0.1% mercuric chloride to disinfect the explants for 10 min. This is because prolonged mercuric chloride treatment damages the explant tissue, especially aggravating cell necrosis on the cut surface, thereby inducing the production and oxidation of more phenolic substances and exacerbating explant browning. The browning rate of Comparative Example 5 was 9 times higher than that of Example 1. Compared with Example 1, Comparative Example 5 did not undergo dark culture for rooting culture, and browning began to appear from the 3rd day after inoculation. The browning rate continued to increase with the extension of culture time, indicating that different light intensities have a significant impact on the browning rate of Litsea cubeba.

[0084] As shown in Table 2, the rooting medium in Example 1 used 1 / 2 WPM as the basic medium, with the addition of IBA 1.0 mg / L, IAA 1.0 mg / L, PVP 1.0 g / L, and activated carbon 1.0 g / L. It was cultured in the dark for 5 days at 23℃, and rooted Litsea cubeba tissue culture seedlings were obtained, with 6 roots and a root length of more than 3 cm. Compared with Comparative Examples 1-5, Example 1 shows that using the method of the present invention for rooting culture, the rooted tissue culture seedlings of Litsea cubeba have the lowest browning rate, the highest rooting rate, and the best number of roots and root length.

[0085] The bud induction effects of stem segments in Comparative Examples 1-5 are shown in the following figures. Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 As shown.

[0086] The rooting results of comparative examples 1-5 are shown below. Figure 23 , Figure 24 , Figure 25 , Figure 26 , Figure 27 As shown.

[0087] pass Figure 3 and Figure 23-27 The comparison shows that Comparative Examples 1-5 exhibited severe browning and yellowing, and a low number of roots, while Example 1 of this application showed a better rooting effect. Figure 3 It has a large root system, green leaves, and a robust plant.

[0088] Table 3. Shoot proliferation during subculture of Example 1 and Comparative Examples 1-5

[0089] As shown in Table 3, the method of this invention was used to subculture *Litsea cubeba* bud clusters by changing the composition and ratio of the subculture medium and the culture conditions to obtain proliferating bud clusters. Compared with Comparative Examples 1-5, Example 1 showed a significantly increased proliferation rate of bud clusters. Some buds began to emerge 7 days after inoculation, and the final proliferation coefficient of the subcultured bud clusters was 8.9. Furthermore, the obtained buds did not exhibit vitrification (e.g., ...). Figure 2 ).

[0090] The following are the subgeneration proliferation effect diagrams for Examples 2-4: Figure 12 , Figure 13 , Figure 14 As shown.

[0091] Comparative Examples 1-5 show the effects of successive proliferation. Figure 15 , Figure 16 , Figure 17 , Figure 18 , Figure 19 .

[0092] The explant disinfection method, hormone type and concentration, low temperature treatment, dark culture, culture conditions, and appropriate addition of anti-browning agents used in this invention can all effectively reduce the browning rate of Litsea cubeba.

[0093] In the process of obtaining Litsea cubeba tissue culture seedlings from Litsea cubeba explants using the method of this invention, compared with the above comparative example, the explants are disinfected with benzalkonium chloride and NaClO to prevent Hg2+ from damaging the explant tissue or aggravating cell necrosis on the cut surface, and to avoid inducing and oxidizing more phenolic substances, thereby reducing explant browning; the formulation of the primary culture medium obtained by the method of this invention can significantly reduce the production of quinone substances in the culture medium, achieving the effect of reducing the browning rate; the subculture medium and culture method of this invention significantly improve the proliferation coefficient of clustered shoots and the quality of subcultured shoots; the rooting medium and culture method of this invention significantly promote root development and improve the rooting rate and rooting quality of Litsea cubeba tissue culture seedlings.

[0094] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for rapid propagation of Litsea cubeba via tissue culture that effectively reduces browning and increases rooting rate, characterized in that, Includes the following steps: (1) Explant collection and disinfection: On a sunny day, collect semi-lignified branches of healthy female and male plants that are free from diseases and pests. Cut the stem segments with axillary buds into 4cm to 6cm lengths as explants; rinse the surface mud and sand with clean water, and then disinfect them. (2) Primary induction culture: Cut off 1 mm from both ends of the explant obtained in step (1) with scissors and trim it into stem segments with 1 to 2 buds. Insert the segments vertically into the prepared primary induction culture medium to obtain the initial buds. (3) Subculture: When the initial shoot height is 3cm to 6cm, cut off the old leaves, retain the terminal bud and part of the petiole, and prune it into a single shoot with a height of 1.0cm to 2.0cm. Transfer it into the prepared subculture culture medium, and inoculate 5 to 8 plants per bottle. After 20 to 25 days of culture, the subculture bud clusters will be formed. When carrying out subculture culture again, cut off the callus tissue at the bottom of the bud clusters and cut it into single shoots or small bud clusters with 2 to 3 buds. Inoculate them into the subculture culture medium, and inoculate 5 to 8 plants per bottle. (4) Rooting culture: Select a single apical bud with a height of ≥1 cm from the subculture buds in step (3), and vertically insert it into the prepared rooting culture medium under sterile conditions. Culture for 15 to 20 days to obtain a robust tissue culture rooted seedling. (5) Hardening and transplanting: When the root length of the tissue culture rooted seedlings in step (4) is ≥1.0 cm and the number of roots is more than 3, the healthy tissue culture rooted seedlings are moved to an outdoor seedling greenhouse for hardening for 5 to 10 days. After that, the root culture medium is washed off with clean water and transplanted into a sterilized substrate seedling container and seedling management is carried out.

2. The method for rapid propagation of Litsea cubeba via tissue culture according to claim 1, which effectively reduces browning and improves rooting rate, is characterized in that: The specific steps of the disinfection treatment described in step (1) are as follows: After cleaning the mud and sand off the surface of the explant, first soak it in a 2-3% volume concentration of detergent for 10-15 minutes with shaking, then soak it in a 2-5% volume concentration of benzalkonium chloride solution for 30-60 minutes, rinse it with running water for 1-2 hours, rinse it with sterile water 3-5 times, then seal it in a sterile culture dish and refrigerate it at 7-9℃ for 10-15 hours. Then, on a clean bench, soak it in a 70-75% volume concentration of alcohol solution for 10-30 seconds, rinse it with sterile water 3-5 times, and finally disinfect it in a 4-6% volume concentration of sodium hypochlorite solution for 25-30 minutes, rinse it with sterile water 3-5 times, place the explant material on sterile filter paper to absorb the moisture, and set it aside for later use.

3. The method for rapid propagation of Litsea cubeba via tissue culture according to claim 1, which effectively reduces browning and increases rooting rate, is characterized in that: The control conditions for the initial induction culture described in step (2) are as follows: dark culture for 5 to 7 days, temperature of 25±3℃, followed by light culture for 8 to 10 days, light intensity of 1600 to 2500 Lx, and light duration of 12 to 14 h / d.

4. The method for rapid propagation of Litsea cubeba via tissue culture according to claim 1, which effectively reduces browning and improves rooting rate, is characterized in that: The stem segment length in step (2) is 1cm to 1.5cm, and the formula of the primary induction medium is: MS, 0.5 to 2.5 mg / L TDZ, 0.1 to 0.5 mg / L NAA, 0.5 to 2.0 mg / L KT, 0.005 to 0.02 mg / L ascorbic acid, 0.5 to 2 g / L PVP, 3 to 5 g / L agar, 28 to 32 g / L sugar, and pH 5.8 to 6.

2.

5. The method for rapid propagation of Litsea cubeba via tissue culture according to claim 1, which effectively reduces browning and improves rooting rate, is characterized in that: The formulation of the subculture proliferation medium in step (3) is as follows: MS, 0.5-2.0 mg / L 6-BA, 0.3-0.5 mg / L NAA, 0.5-3.0 mg / L KT, 3-5 g / L agar, 28-32 g / L sugar, 0.01-0.02 mg / L riboflavin, 0.005-0.02 mg / L ascorbic acid, 0.5-2 g / L Flower Treasure No. 1, pH 5.8-6.

2.

6. The method for rapid propagation of Litsea cubeba via tissue culture according to claim 1, which effectively reduces browning and improves rooting rate, is characterized in that... In step (3), the controlled conditions for cultivation are: temperature 25±3℃, light intensity 2500~9000 Lx, and light duration 12~16 h / d.

7. The method for rapid propagation of Litsea cubeba via tissue culture according to claim 1, which effectively reduces browning and improves rooting rate, is characterized in that: The rooting medium in step (4) is formulated as follows: 1 / 2 WPM, 1-1.5 mg / L IBA, 0.5-1.0 mg / L IAA, 1-2.0 g / L activated carbon, 0.5-2 g / L PVP, 3-5 g / L agar, 14-16 g / L sugar, and pH 5.8-6.

2.

8. The method for rapid propagation of Litsea cubeba via tissue culture according to claim 1, which effectively reduces browning and improves rooting rate, is characterized in that... In step (4), the culture control conditions are as follows: dark culture for 5 days, followed by light culture for 10 to 15 days, with a temperature of 25±3℃, a light intensity of 2500 to 9000 Lx, and a light duration of 12 to 16 h / d.

9. The method for rapid propagation of Litsea cubeba via tissue culture according to claim 1, which effectively reduces browning and increases rooting rate, is characterized in that... In step (5), the seedling management method is as follows: place the seedlings in a nursery with a shade net and spray water once or twice a day, morning and evening; after 7 days, spray with a compound fertilizer solution with a concentration of 0.3% to 0.5% once or twice every 10 days; within one week after transplanting, maintain the light transmittance at 40% to 50%, gradually increase the light after one week, and increase the light transmittance to 80% to 90% one month after transplanting.

10. The method for rapid propagation of Litsea cubeba via tissue culture according to claim 1, which effectively reduces browning and increases rooting rate, is characterized in that... Three days before transplanting the seedlings described in step (5), the seedling substrate is thoroughly disinfected by rinsing with a potassium permanganate solution with a volume concentration of 0.5-1.0%. One day later, the potassium permanganate solution is rinsed off with clean water.

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