Tissue culture and rapid propagation method for castanopsis sclerophylla

Abstract

The invention belongs to the technical field of plant tissue culture, and relates to a tissue culture and rapid propagation method for castanopsis sclerophylla. According to the method, castanopsis sclerophylla seeds are used as materials, and through sterilization method comparison, basic culture medium screening, hormone proportioning optimization and the like, an efficient tissue culture and rapid propagation technical system of castanopsis sclerophylla is established through seed sterile germination, bud proliferation, strong bud rooting and acclimatization and transplantation, and complete tissue culture progeny plants are formed. 100.0% of the seeds in the system germinate and sprout; the propagation coefficient of buds is large and reaches 6.8; the rooting rate reaches 92.8%, the root system is developed, the average is 4.1 roots per plant, the root length is 4.6 cm, and lateral roots are dense; the transplanting survival rate is high and can reach 95.2% or above; and the method is not limited by factors such as seasons, climate and regions, and can realize annual efficient and rapid propagation. According to the method, the blank of tissue culture and rapid propagation of the castanopsis sclerophylla is filled, a stable and efficient regeneration method for the in-vitro culture plant of the castanopsis sclerophylla is provided, large-scale seedling culture can be carried out, the cultured castanopsis sclerophylla seedlings are robust, neat and consistent, and a high-quality seedling guarantee is provided for improving the economic benefit of an artificial forest of the castanopsis sclerophylla.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a rapid propagation method for Castanopsis fargesii tissue culture based on aseptic seed germination, bud proliferation, strong bud rooting, and seedling hardening and transplanting. Background Technology

[0002] bitter oak ( Castanopsis sclerophylla (Lindl.) Schott., also known as bitter chestnut, oak chestnut, or bitter oak, belongs to the genus *Lindl.* of the family Fagaceae. Castanopsis A type of evergreen tree, reaching 15-20 meters in height and up to 50 cm in diameter at breast height, *Castanopsis fargesii* is found south of the Yangtze River and north of the Five Ridges, including Zhejiang, Jiangxi, Hunan, Fujian, Yunnan, and Guangdong provinces. It is a national second-class protected plant. *Castanopsis fargesii* prefers sunlight, is drought-tolerant, has abundant branches and leaves, a long lifespan, and strong resistance to disease. It is an important species for building evergreen broad-leaved forests in subtropical regions, playing a certain role in controlling the composition, appearance, and function of the community. *Castanopsis fargesii* has excellent fire resistance and is often mixed with other major timber species in southern China, such as Chinese fir and Masson pine, to create mixed coniferous and broad-leaved forests, providing both fire-retardant effects and improving forest quality. *Castanopsis fargesii* grows relatively quickly, has a wide crown, beautiful tree shape, and strong dust-trapping and noise-reducing abilities, making it suitable for planting as a street tree and an excellent species for urban and courtyard greening.

[0003] The bitter oak (Castanopsis spp.) has excellent wood properties. Its wood is yellow or yellowish-white, dense in structure, straight in grain, and elastic. It is light to medium in strength, low to medium in toughness, and has high to medium shrinkage with moderate to high differential shrinkage. It is moisture-resistant and rot-resistant, making it excellent for high-quality construction, bridges, furniture, and sports equipment. It is also a good material for cultivating edible fungi. The bark of the bitter oak contains high-purity tannins, which can be used to extract tannin. Fresh bitter oak leaves contain a large amount of crude protein and can be used as livestock feed. The kernels of the bitter oak are a natural food with both medicinal and edible uses. According to the *Compendium of Materia Medica*, bitter oak kernels have the effects of improving eyesight, relieving hunger, invigorating qi, strengthening muscles and bones, strengthening the waist and knees, and replenishing deficiencies. They are edible and are the raw material for making traditional delicacies such as bitter oak tofu, making them an important source of forest food. They also have medicinal uses; the flavonoids extracted from them can treat cardiovascular diseases and diabetes, and lower blood lipids, demonstrating high medicinal value. In recent years, due to continuous logging and habitat destruction, the natural wild resources of Castanopsis sclerophylla have been declining. With the increasing social demand for Castanopsis sclerophylla, large-scale production of its seedlings is particularly important. Currently, the seedlings used in Castanopsis sclerophylla plantations are mostly produced using mixed seed collection, resulting in significant individual differentiation and unevenness in seedlings and the resulting stands, which in turn affects the quality, yield, and economic benefits of the plantations.

[0004] In summary, although the Pinus kesiya has important ecological, economic and medicinal value, its natural population is facing degradation, and artificial forest seedling is mostly dependent on seedling, leading to unstable genetic traits of forest trees, inconsistent growth, and low survival rate of afforestation. There is no systematic research and report on efficient rapid propagation of Pinus kesiya through tissue culture in the existing literature. Therefore, it is urgent to establish a stable, efficient and scalable tissue culture and rapid propagation technology of Pinus kesiya. SUMMARY

[0005] The purpose of the present application is to provide a tissue culture and rapid propagation method of Pinus kesiya.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows: a tissue culture and rapid propagation method of Pinus kesiya, comprising the following steps: (1) Seed aseptic germination: take mature Pinus kesiya fruits, remove the pericarp, wash the seeds with washing powder water, rinse with running water for 20-30 min, transfer to a clean bench, soak in 75% alcohol solution for 50-70 s, wash with sterile water for 2-3 times, soak in 0.1% mercury solution for 18-22 min, wash with sterile water for 4-5 times, and dry the surface water with sterile filter paper. Cut open the seed coat, take out the embryo and inoculate on the seed aseptic germination medium for 25-35 days; The formula of the seed aseptic germination medium is: WPM medium + 25.0-35.0 g / L sucrose + 6.5-7.5 g / L agar; (2) Bud proliferation culture: take the buds formed by seed germination, inoculate on the bud proliferation culture medium for 30-40 days to proliferate into cluster buds; The formula of the bud proliferation culture medium is: WPM medium + 1.8-2.2 mg / L 6-BA + 0.4-0.6 mg / L TDZ + 25.0-35.0 g / L sucrose + 6.5-7.5 g / L agar; (3) Strong bud and rooting culture: divide the proliferated cluster buds into single buds, inoculate in the strong bud and rooting culture medium according to the polarity for 30-40 days; The formula of the strong bud and rooting culture medium is: 1 / 2 WPM + 0.4-0.6 mg / L TDZ + 0.8-1.2 mg / L IBA + 25-35 g / L sucrose + 6.5-7.5 g / L agar; (4) Seedling training and transplanting: place the rooted bottle seedlings in a light incubator with a light intensity of 60-100 µmol·m -2 ·s -1The aseptic seedling is sealed for 2-3 days under scattered light, and is opened for 1-2 days, and the temperature is controlled at 15-30 DEG C. The aseptic seedling is taken out, washed, soaked with 50% carbendazim wettable powder 800-1200 times liquid for 30-60 s, and dried, and is transplanted into a seedling container, the substrate covers the root system, is slightly pressed, and is poured with a root fixing water. The substrate is mixed by peat soil and perlite at a volume ratio of 3:1.

[0007] After transplanting, the film is covered for moisturizing, the film is uncovered after 12-15 days, the substrate is kept moist, the air relative humidity is 85-95%, the culture temperature is controlled at 15-30 DEG C, and the shading degree is 65-75%. After 25-30 days of transplanting, 1-2% complex fertilizer in mass and volume percentage is sprayed, and then is sprayed every 12-15 days. After new buds and new roots grow on the seedling, the light intensity is gradually increased until full light, and the seedling is transplanted when the height reaches 18-20 cm.

[0008] The culture conditions of steps (1), (2) and (3) are as follows: temperature 23-27 DEG C, light intensity 2000-2500 Lx, and light and dark alternation culture is carried out for 12-14 h per day.

[0009] The pH value of the culture medium used in steps (1), (2) and (3) is 5.7-5.9.

[0010] The formula of the aseptic germination culture medium of the seed is as follows: WPM culture medium + 25.0-35.0 g / L sucrose + 6.5-7.5 g / L agar.

[0011] The formula of the bud proliferation culture medium is as follows: WPM culture medium + 1.8-2.2 mg / L 6-BA + 0.4-0.6 mg / L TDZ + 25.0-35.0 g / L sucrose + 6.5-7.5 g / L agar.

[0012] The formula of the strong bud and rooting culture medium is as follows: 1 / 2 WPM + 0.4-0.6 mg / L TDZ + 0.8-1.2 mg / L IBA + 25-35 g / L sucrose + 6.5-7.5 g / L agar.

[0013] The beneficial technical effects of the application are as follows: Compared with existing technologies, this invention provides a rapid propagation method for Castanopsis sclerophylla through tissue culture. Through aseptic seed germination, bud proliferation culture, bud strengthening and rooting culture, as well as hardening and transplanting, a highly efficient rapid propagation technology system for Castanopsis sclerophylla through tissue culture is established, forming complete tissue-cultured progeny plants. This system achieves 100% seed germination; the bud proliferation coefficient is high, reaching 6.8; the rooting rate reaches 92.8%, with a well-developed root system, an average of 4.1 adventitious roots per plant, a root length of 4.6 cm, and dense lateral roots; and the transplant survival rate is high, reaching over 95.2%. The cultivation process is not limited by season, climate, or region, enabling efficient and rapid propagation throughout the year. This invention fills the gap in rapid propagation of Castanopsis sclerophylla through tissue culture, providing a stable and efficient method for the regeneration of Castanopsis sclerophylla plants from in vitro culture. It allows for large-scale seedling production, producing robust and uniform Castanopsis sclerophylla seedlings, providing a high-quality seedling guarantee for improving the economic benefits of Castanopsis sclerophylla plantations. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 It is the mature bitter oak fruit of Example 1.

[0016] Figure 2 It is the embryo after sterilization and removal of the seed coat in Example 1.

[0017] Figure 3 This is Example 1, where the seed germinates to form a bud and root.

[0018] Figure 4 Example 2 shows how to induce cluster buds from the buds formed by seed germination to achieve bud proliferation.

[0019] Figure 5 In Example 3, the regenerated shoots elongate while roots are induced to form.

[0020] Figure 6 The regenerated seedlings in Example 3 developed a well-developed root system. Detailed Implementation

[0021] The present invention will be further described below with reference to specific implementation schemes. These embodiments should be understood as merely preferred examples of the present invention and not as limiting the present invention in any way.

[0022] Example 1: The impact of aseptic seed germination method on the present invention 1) Experimental materials: Mature bitter oak fruits were harvested from healthy plants in November. Figure 1 This is used for aseptic seed germination culture. The culture medium is prepared using commercially available basic culture medium, hormones, sucrose, and agar.

[0023] 2) Experimental methods: Remove the pericarp, select seeds with intact seed coats, rinse with detergent water and then rinse under running water for 20-30 minutes. Sterilize the seeds using different sterilization treatments, and then blot dry the surface moisture with sterile filter paper for later use.

[0024] The aseptic germination medium for seeds was based on MS and WPM, with or without added hormones, with 30 g / L sucrose and 7 g / L agar added. The pH was adjusted to 5.8 and then sterilized at 121 °C for 20 min. After cooling and solidification, the medium was ready for use.

[0025] Sterilized and seed coat removed embryos ( Figure 2 The culture medium was inoculated onto sterile germination medium and placed on a culture rack in a culture room for germination culture. The culture room temperature was 23 ℃~27 ℃, the light intensity at the bottom of the culture layer was 2000~2500 Lx, and the light duration was 12~14 h per day.

[0026] Observe and record seed germination and growth, and evaluate the effects of sterilization methods and culture media on seed germination and growth. After 15 days of inoculation, calculate the seed contamination rate and germination rate. Contamination rate (%) = (number of contaminated seeds / total number of inoculated seeds) × 100; Germination rate (%) = (number of germinated seeds / total number of inoculated seeds) × 100.

[0027] 3) Experimental Results and Conclusions: After the embryos are inoculated onto the germination medium, they absorb water and swell, and begin to germinate in 2-3 days. By 30 days, roots and shoots have formed. Figure 3Seed contamination rate and germination rate were recorded 15 days after inoculation. Table 1 shows that the sterilization treatment method affected the seed contamination rate and germination rate. Treatment ①: The seed coat was removed first, then the seeds were soaked in 75% alcohol for 40 seconds, rinsed 2-3 times with sterile water, then soaked in 0.1% mercuric chloride for 7 minutes, and rinsed 5-6 times with sterile water. The contamination rate and germination rate of the treated seeds after inoculation were 23.7% and 76.3%, respectively. Treatment ②: The seeds were soaked in 75% alcohol for 40 seconds, rinsed 2-3 times with sterile water, then soaked in 0.1% mercuric chloride for 12 minutes, and rinsed 5-6 times with sterile water. The seed coat was then removed, and the seeds were soaked in 0.1% mercuric chloride for 6 minutes, and rinsed 5-6 times with sterile water. The contamination rate and germination rate of the treated seeds after inoculation were 16.5% and 83.5%, respectively. Compared with treatment ①, treatment ② showed a lower seed contamination rate and a higher germination rate. However, treatments ① and ② both involved soaking and rinsing after removing the seed coat, which easily damaged the seed structure, resulting in about half of the seeds germinating with abnormal buds or roots. In treatments ③, ④, and ⑤, the seeds were first soaked in 75% alcohol for 60 seconds, rinsed 2-3 times with sterile water, then soaked in 0.1% mercuric chloride for varying durations, rinsed 5-6 times with sterile water, and then inoculated after removing the seed coat. As the mercuric chloride soaking time increased, the contamination rate after seed inoculation gradually decreased, and the germination rate gradually increased. Treatment ⑤, soaked in 0.1% mercuric chloride for 20 minutes, resulted in uncontaminated seeds with 100.00% germination, and both buds and roots were normal.

[0028] Table 1. Effects of different sterilization treatments on the aseptic germination of Castanopsis fargesii seeds

[0029] As shown in Table 2, the seeds could germinate at a 100.0% rate on all three culture media, but the seedlings were in different states. The buds that germinated on culture medium ① MS were normal, the buds that germinated on culture medium ② WPM were robust and grew quickly, and the buds that germinated on culture medium ③ were thinner and weaker.

[0030] Table 2. Effects of different culture media on the aseptic germination of Castanopsis fargesii seeds

[0031] In summary, the appropriate sterilization method for Castanopsis sclerophylla seeds is to first soak them in 75% alcohol for 60 seconds, rinse them 2-3 times with sterile water, then soak them in 0.1% mercuric chloride for 20 minutes, rinse them 5-6 times with sterile water, and finally remove the seed coat. WPM basal medium without any added hormones is a suitable medium for the aseptic germination of Castanopsis sclerophylla seeds.

[0032] Example 2: The impact of bud proliferation culture method on the present invention 1) Experimental materials: The plant material used in the experiment consisted of aseptic seedling shoots that germinated from Castanopsis fargesii seeds 25–35 days after inoculation. The culture medium was prepared using commercially available basic culture medium, hormones, sucrose, and agar.

[0033] 2) Experimental methods: The bud proliferation medium was based on WPM, with different types and concentrations of hormones added, along with 30 g / L sucrose and 7 g / L agar. After adjusting the pH to 5.8, the medium was sterilized at 121 °C for 20 min and cooled to solidify before use.

[0034] Seedlings formed from aseptic germination of seeds are picked up with tweezers, and buds are cut off and inoculated onto bud proliferation medium according to polarity. They are then placed on culture racks in a culture room and cultured for 30–40 days to induce the formation of bud clusters. The culture room temperature is 23 ℃–27 ℃, the light intensity at the bottom of the culture layer is 2000–2500 Lx, and the light duration is 12–14 h per day.

[0035] Observe and record the proliferation of buds, and calculate the proliferation ratio.

[0036] 3) Experimental Results and Conclusions: Buds cut from sterile seedlings are inoculated and cultured. The base of the bud gradually swells, and subsequently, clusters of buds form on the swollen part or stem. Figure 4 Table 3 shows that the type and concentration of hormones affect bud proliferation. The induction effect of using 6-BA alone is not as good as that of using 6-BA and TDZ simultaneously; the former resulted in a proliferation ratio of 3.5–4.8, while the latter ranged from 5.2 to 6.8. The highest bud proliferation ratio, reaching 6.8, was observed on medium ⑤. Therefore, WPM medium supplemented with 2.0 mg / L 6-BA and 0.5 mg / L TDZ is a suitable medium for the proliferation of Castanopsis sclerophylla buds.

[0037] Table 3. Effects of different culture media on the proliferation of Castanopsis sclerophylla shoots

[0038] Example 3: The impact of bud strengthening and rooting culture methods on the present invention 1) Experimental materials: The plant material used in the experiment was shoot clusters formed through propagation culture. The culture medium was prepared using commercially available basic culture medium, hormones, sucrose, and agar.

[0039] 2) Experimental methods: For bud strengthening and rooting culture, WPM or 1 / 2 WPM was used as the basic medium, with different types and concentrations of hormones added, along with 30 g / L sucrose and 7 g / L agar. After adjusting the pH to 5.8, the culture was sterilized at 121 °C for 20 min and cooled to solidify for later use.

[0040] The proliferating bud clusters were cut into individual buds and inoculated into bud-strengthening and rooting media according to polarity. The culture bottles were placed on the culture rack in the culture room and cultured for 30–40 days to induce rooting while strengthening the buds. The culture room temperature was 23 ℃–27 ℃, the light intensity at the bottom of the culture layer was 2000–2500 Lx, and the light duration was 12–14 h per day.

[0041] Observe the growth and rooting of the buds, and calculate the rooting rate, number of adventitious roots, and root length. Rooting rate (%) = number of rooted buds / total number of inoculated buds × 100.

[0042] 3) Experimental Results and Conclusions: After the bud clusters were cut into single buds, they were inoculated onto a bud strengthening and rooting medium and cultured. The buds gradually elongated, and white adventitious roots grew from the base of the buds. Figure 5 As the cultivation time increases, the adventitious roots darken in color and develop lateral roots. Figure 6 Table 4 shows that the type of basic culture medium, the type and concentration of hormones affect the bud development and rooting of Castanopsis fargesii. On 1 / 2 WPM medium supplemented with 1.0 mg / L NAA, IAA, or IBA alone, the rooting rate was 85.8–88.7%, with 2.1–3.5 roots per plant and a root length of 2.8–3.9 cm, and some buds showed browning. On media supplemented with 0.5 mg / L TDZ and 1.0 mg / L IBA, the browning of buds was reduced. Few lateral roots formed on medium ④, which used WPM as the basic medium. However, many lateral roots formed on medium ⑤, which used 1 / 2 WPM as the basic medium, and the buds were robust, with a rooting rate reaching 92.8%, an average of 4.1 adventitious roots per plant, and an average root length of 4.6 cm. Therefore, 1 / 2 WPM medium supplemented with 0.5 mg / L TDZ and 1.0 mg / L IBA is a suitable medium for promoting shoot growth and root development in Castanopsis fargesii.

[0043] Table 4. Effects of different culture media on shoot growth and root development of Castanopsis fargesii.

[0044] The embodiments described above can be further combined or replaced, and these embodiments are merely descriptions of preferred embodiments of the present invention, not limitations on the concept and scope of the present invention. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the inventive concept are all within the protection scope of the present invention. The protection scope of the present invention is given by the appended claims and any equivalents.

Claims

1. A method for rapid propagation of Castanopsis sclerophylla through tissue culture, characterized by the following steps: include: (1) Aseptic germination of seeds: Take mature bitter oak fruit, remove the pericarp, wash with detergent water and rinse with running water, then soak in 75% alcohol for 50-70 seconds, rinse with sterile water 2-3 times, soak in 0.1% mercuric chloride solution for 18-22 minutes, rinse with sterile water 4-5 times, dry the surface water, cut open the seed coat to take out the embryo, and inoculate it on the aseptic germination medium for 25-35 days; The aseptic germination medium for the seeds is WPM medium + 25.0–35.0 g / L sucrose + 6.5–7.5 g / L agar; (2) Bud proliferation culture: The buds that germinated in step (1) were inoculated on the bud proliferation culture medium and cultured for 30 to 40 days to induce the formation of cluster buds; The bud proliferation medium consisted of WPM medium + 1.8–2.2 mg / L 6-BA + 0.4–0.6 mg / L TDZ + 25.0–35.0 g / L sucrose + 6.5–7.5 g / L agar; (3) Bud strengthening and rooting culture: The cluster of buds formed in step (2) is divided into single buds and inoculated into bud strengthening and rooting culture medium according to polarity and cultured for 30 to 40 days; The bud-strengthening and rooting medium consisted of 1 / 2 WPM medium + 0.4–0.6 mg / L TDZ + 0.8–1.2 mg / L IBA + 25–35 g / L sucrose + 6.5–7.5 g / L agar; (4) Hardening off and transplanting: Rooting bottle seedlings were hardened off and transplanted at 60–100 μmol·m⁻² ... -2 ·s -1 Harden off the seedlings under diffused light for 2-3 days with the top closed and 1-2 days with the bottom open, at a temperature of 15-30°C. After removing the seedlings, wash the roots and soak them in a 50% carbendazim solution diluted 800-1200 times for 30-60 seconds, then let them air dry. Transplant them into a substrate made of peat moss and perlite in a 3:1 volume ratio, water thoroughly, cover with a thin film to retain moisture, and uncover the film after 12-15 days. Keep the substrate moist, maintain a relative humidity of 85-95%, a temperature of 15-30°C, and a shading level of 65%-75%. Spray with 1-2‰ compound fertilizer 25-30 days after transplanting, once every 12-15 days. After the seedlings have grown new buds and roots, gradually increase the light exposure to full sunlight. When the seedlings are 18-20 cm tall, transplant them to the nursery.

2. The method according to claim 1, characterized in that, The pH value of the seed sterile germination medium described in step (1) is 5.7 to 5.

9.

3. The method according to claim 1, characterized in that, The pH value of the bud proliferation medium described in step (2) is 5.7 to 5.

9.

4. The method according to claim 1, characterized in that, The pH value of the bud-strengthening and rooting culture medium described in step (3) is 5.7 to 5.

9.

5. The method according to claim 1, characterized in that, The culture conditions for steps (1), (2) and (3) are: temperature 23-27°C, light intensity 2000-2500 Lx, light exposure 12-14 h per day, and alternating light and dark culture.

6. The method according to claim 1, characterized in that, The sterilization process described in step (1) includes: First, soak in 75% alcohol for 60 seconds, rinse with sterile water 2-3 times, then soak in 0.1% mercuric chloride solution for 20 minutes, rinse with sterile water 5-6 times, and then remove the seed coat.

7. The method according to claim 1, characterized in that, The bud proliferation medium in step (2) preferably contains 2.0 mg / L 6-BA and 0.5 mg / L TDZ.

8. The method according to claim 1, characterized in that, The bud-strengthening and rooting culture medium described in step (3) preferably contains 0.5 mg / L TDZ and 1.0 mg / L IBA.

9. The method according to claim 1, characterized in that, During the growth process of seedlings after transplanting and before they leave the nursery, the relative humidity of the air should be controlled at 85-95%, the shading degree at 65-75%, and the temperature at 15-30℃.

10. The method according to claim 1, characterized in that, It can achieve a survival rate of ≥ 95% for Castanopsis sclerophylla seedlings, and is not limited by season, climate or region, enabling efficient and rapid propagation throughout the year.

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