In-vitro rapid propagation method for polygonum multiflorum

Abstract

The invention discloses a tissue culture and rapid propagation method of polygonum multiflorum. The method comprises the steps of selection and disinfection of explants, primary induction culture, subculture and rooting culture. According to the method, the tender stem tip of the polygonum multiflorum is selected as the explant, and the explant has the advantages of being easy to disinfect and easy to differentiate. Disinfecting the explant with a sodium hypochlorite solution, and establishing a sterile system; meanwhile, by using the primary culture medium, the stem tip is induced to grow into a plant quickly; then multiple shoots are formed on a proliferation culture medium, few calluses exist at bud bases, variation is reduced, and the proliferation coefficient is high; by means of the rapid propagation method, the bud propagation coefficient and the rooting rate can be increased, and the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to the field of rapid propagation of horticultural plant tissue culture, and particularly relates to a method for in vitro rapid propagation of Panax multiflorum Thunb. var. multiflorum (Thunb.) Hand.-Mazz. × Panax ginseng C. A. Mey. Background Art

[0002] Panax multiflorum Thunb. var. multiflorum (Thunb.) Hand.-Mazz. × Panax ginseng C. A. Mey. originates from Taiwan Province, China. It is a new species hybridized from Polygonum multiflorum Thunb. and Panax ginseng C. A. Mey. The test results of the Institute of Biotechnology, National Chung Hsing University in Taiwan Province, China show that Panax multiflorum Thunb. var. multiflorum (Thunb.) Hand.-Mazz. × Panax ginseng C. A. Mey. only contains trace amounts of emodin, and many components are higher than those of red-fleshed and white-fleshed Polygonum multiflorum Thunb. It is a new medicinal material with extremely high medicinal value, having the effects of tonifying essence and blood, detoxifying and arresting malaria, and moistening the intestines and relieving constipation. Moreover, it also has good curative effects on symptoms such as anemia, neurasthenia, white hair, and hair loss.

[0003] In the prior art, the main propagation method of Panax multiflorum Thunb. var. multiflorum (Thunb.) Hand.-Mazz. × Panax ginseng C. A. Mey. is cuttage propagation of fleshy roots, which has problems such as a long growth cycle and being restricted by seasons, with a relatively low propagation coefficient and unable to meet the demand for seedlings in large-scale planting production. Tissue culture technology has the advantages of being unaffected by the external environment, high propagation coefficient, and low production cost. At present, there is no report on the tissue culture rapid propagation of Panax multiflorum Thunb. var. multiflorum (Thunb.) Hand.-Mazz. × Panax ginseng C. A. Mey. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for in vitro rapid propagation of Panax multiflorum Thunb. var. multiflorum (Thunb.) Hand.-Mazz. × Panax ginseng C. A. Mey., aiming to improve the bud proliferation coefficient and rooting rate of Panax multiflorum Thunb. var. multiflorum (Thunb.) Hand.-Mazz. × Panax ginseng C. A. Mey. and reduce the production cost.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: A method for in vitro rapid propagation of Panax multiflorum Thunb. var. multiflorum (Thunb.) Hand.-Mazz. × Panax ginseng C. A. Mey. comprises the following steps: (1) Selection and disinfection of explants: Select the young shoot tips of Panax multiflorum Thunb. var. multiflorum (Thunb.) Hand.-Mazz. × Panax ginseng C. A. Mey. about 1 - 1.5 cm long as explants, soak them in saturated washing powder solution for 10 min, rinse them with running water and then soak them in 75% w / v alcohol for 30 s, rinse them 3 times with sterile double-distilled water, and then soak them in 3% w / v sodium hypochlorite solution for 10 min, shaking the bottle constantly during this period. After completion, rinse them 5 times with sterile double-distilled water; (2) Primary culture of explants: Inoculate the explants obtained in step (1) into an MS medium containing 6-BA and IBA for primary induction culture; (3) Subculture proliferation culture of explants: Cut the plants obtained from the primary induction culture in step (2) into single small buds or stem segments 1 - 1.5 cm long, inoculate them into an MS medium containing 6-BA and IBA for subculture, and grow into cluster buds; (4) Rooting culture of explants: When the bud seedlings of the cluster buds grow to 2 - 4 cm, inoculate them into a rooting medium and culture until they take root and become seedlings.

[0006] Furthermore, in step (1), the Panax multiflorum Thunb. var. multiflorum (Thunb.) Hand.-Mazz. × Panax ginseng C. A. Mey. is obtained by hybridizing Polygonum multiflorum Thunb. and Panax ginseng C. A. Mey.

[0007] Further, in step (2), the concentration of 6-BA is 1.5 mg / L, and the concentration of IBA is 0.3 mg / L.

[0008] Further, in step (2), the temperature for primary induction culture is 25 ± 2 °C, the humidity is 100%, the light intensity is 3000 - 5000 Lux, the light time is 12 - 14 hours, and the induction culture time is 25 - 35 days.

[0009] Further, in step (3), the concentration of 6-BA is 1.5 mg / L, and the concentration of IBA is 0.1 mg / L.

[0010] Further, in step (3), the temperature for subculture proliferation culture is 25 ± 2 °C, the humidity is 100%, the light intensity is 3000 - 5000 Lux, the light time is 12 - 14 hours, and the induction culture time is 25 - 35 days.

[0011] Further, in step (3), the subculture proliferation coefficient is 9 - 10.

[0012] Further, in step (4), the rooting medium is 1 / 2MS rooting medium, the temperature for rooting culture is 25 ± 2 °C, the humidity is 100%, the light intensity is 3000 - 5000 Lux, the light time is 12 - 14 hours, and the culture time is 25 - 35 days.

[0013] The advantages of the present invention are as follows: (1) The object of the present invention is Panax multiflorum Thunb. var. multiflorum Thunb. f. hirsutus Hoo & Tsai, which is from Taiwan Province, China, and is a new hybrid species. There is no report on the rapid propagation of Panax multiflorum Thunb. var. multiflorum Thunb. f. hirsutus Hoo & Tsai so far.

[0014] (2) The explant of the present invention is the young shoot with 4 - 5 expanded leaves of Panax multiflorum Thunb. var. multiflorum Thunb. f. hirsutus Hoo & Tsai. The young shoot tip about 1 - 1.5 cm long is selected as the explant. The induction and differentiation growth ability of the shoot tip is stronger than that of the stem segment, effectively improving the disinfection efficiency and the induction rate of primary plants.

[0015] (3) The present invention adopts appropriate primary induction, subculture proliferation culture and strong seedling rooting media, with fast growth, short cycle, less callus, improving the bud proliferation coefficient and rooting rate of Panax multiflorum Thunb. var. multiflorum Thunb. f. hirsutus Hoo & Tsai, and reducing the production cost. Description of the Drawings

[0016] Figure 1 This is the plant formed after 30 days of primary culture of Panax multiflorum Thunb. var. multiflorum Thunb. f. hirsutus Hoo & Tsai in Example 1 of the present invention.

[0017] Figure 2 This is the cluster buds obtained by subculture proliferation of Panax multiflorum Thunb. var. multiflorum Thunb. f. hirsutus Hoo & Tsai in Example 1 of the present invention.

[0018] Figure 3This is the rooted seedling obtained from the rooting culture of ginseng-polygonum multiflorum in Example 1 of the present invention. Detailed implementation mode

[0019] In order to make the objectives, technical solutions and advantages of the present invention clearer and easier to understand, the present invention will be further described in detail below in conjunction with embodiments. It can be understood that the specific implementations described herein are only used to explain the present invention and are not used to limit the present invention.

[0020] The polygonum multiflorum ginseng in the following embodiments is from Taiwan Province, China, and is a new species hybridized from polygonum multiflorum and ginseng flower.

[0021] Example 1 A method for rapid in vitro propagation of polygonum multiflorum ginseng, the method comprising the following steps: (1) Selection and disinfection of explants: Select the young shoot tips with 4-5 expanded leaves of polygonum multiflorum ginseng branches as explants, remove the leaves, cut 1-1.5 cm shoot tips as explants, soak them in saturated washing powder water for 10 min, then rinse with running water for 2 h, and the subsequent steps are completed in a super clean bench; soak the above-cleaned shoot tips in 75% w / v alcohol for 30 s, rinse with sterile double distilled water 3 times, then soak them in 3% w / v sodium hypochlorite solution for 10 min, shake the bottle constantly during this period, and after completion, rinse with sterile double distilled water 5 times, and the sterilization rate reaches 71.95%; (2) Primary culture of explants: Inoculate the explants treated in step (1) into an MS medium containing 1.5 mg / L 6-BA and 0.3 mg / L IBA for primary induction culture, and culture for 30 days under preset temperature, humidity and light culture conditions to form plants (as Figure 1 shown), the induction rate is 96.67%, and the proliferation coefficient reaches 3.9. The temperature of the primary induction culture is 25±2 °C, the humidity is 100%, the light intensity is 5000 Lux, and the light time is 12 hours.

[0022] (3) Subculture proliferation culture of explants: Cut the plants obtained from the primary induction culture into single small buds or stem segments 1-1.5 cm long, and inoculate them into an MS medium containing 1.5 mg / L 6-BA and 0.1 mg / L IBA for subculture for 30 days ( Figure 2 ), culture for 30 days to form clustered buds, and the proliferation coefficient reaches 9.67; the temperature of the subculture is 25±2 °C, the humidity is 100%, the light intensity is 5000 Lux, and the light time is 12 hours.

[0023] (4) Rooting culture of explants: When the bud seedlings grow to about 3 cm, inoculate them into a 1 / 2MS rooting medium for rooting culture for 30 days until they take root and become seedlings ( Figure 3), the rooting rate can reach 100%. The temperature for rooting culture is 25±2°C, the humidity is 100%, the light intensity is 5000 Lux, and the light duration is 12 hours.

[0024] Comparative Example 1 This method is basically the same as that of Example 1, except that in the subculture proliferation culture of the explant, the plants obtained from the primary induction culture are cut into single small buds or stem segments 1-1.5 cm long and then inoculated into an MS medium containing 1.0 mg / L 6-BA and 0.05 mg / L NAA for subculture.

[0025] Comparative Example 2 This method is basically the same as that of Example 1, except that in the subculture proliferation culture of the explant, the plants obtained from the primary induction culture are cut into single small buds or stem segments 1-1.5 cm long and then inoculated into an MS medium containing 1.5 mg / L 6-BA and 0.1 mg / L NAA for subculture.

[0026] The effects of different hormone treatments on the subculture proliferation of Polygonum multiflorum Thunb. var. multiflorum Thunb. are shown in Table 1.

[0027] Table 1 Effects of different hormone treatments on the subculture proliferation of Polygonum multiflorum Thunb. var. multiflorum Thunb. The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. A method for rapid in vitro propagation of Radix Polygoni Multiflori, characterized in that: The steps include: (1) Selection and disinfection of explants: Select young and tender stem tips of Polygonum multiflorum about 1-1.5 cm long as explants, soak them in saturated detergent water for 10 min, rinse them with running water, soak them in 75% w / v alcohol for 30 s, rinse them with sterile double distilled water for 3 times, and then soak them in 3% w / v sodium hypochlorite solution for 10 min, shaking the bottle constantly during the process. After completion, rinse them with sterile double distilled water for 5 times; (2) Primary culture of explants: the explants obtained in step (1) are inoculated into MS medium containing 6-BA and IBA for primary induction culture; (3) Subculture and proliferation of explants: the plants obtained from the primary induction culture in step (2) are cut into single buds or stem segments with a length of 1-1.5 cm, and inoculated into MS medium containing 6-BA and IBA for subculture to grow into clustered buds; (4) Rooting culture of explants: When the buds of clustered buds grow to 2-4 cm, they are inoculated into rooting medium and cultured until they take root and become seedlings.

2. The in vitro rapid propagation method of Radix Polygoni Multiflori according to claim 1, characterized in that: The ginseng and Polygonum multiflorum described in step (1) is obtained by hybridizing Polygonum multiflorum and ginseng flower.

3. The in vitro rapid propagation method of Radix Polygoni Multiflori according to claim 1, characterized in that: In step (2), the concentration of 6-BA is 1.5 mg / L, and the concentration of IBA is 0.3 mg / L.

4. The in vitro rapid propagation method of Radix Polygoni Multiflori according to claim 1, characterized in that: The temperature of the primary induction culture in step (2) is 25±2°C, the humidity is 100%, the light intensity is 3000-5000 Lux, the light time is 12-14 hours, and the induction culture time is 25-35 days.

5. The in vitro rapid propagation method of Radix Polygoni Multiflori according to claim 1, characterized in that: In step (3), the concentration of 6-BA is 1.5 mg / L, and the concentration of IBA is 0.1 mg / L.

6. The in vitro rapid propagation method of Radix Polygoni Multiflori according to claim 1, characterized in that: In step (3), the temperature of the subculture proliferation culture is 25±2°C, the humidity is 100%, the light intensity is 3000-5000 Lux, the light duration is 12-14 hours, and the induction culture duration is 25-35 days.

7. The in vitro rapid propagation method of Radix Polygoni Multiflori according to claim 1, characterized in that: The proliferation coefficient of the subculture in step (3) is 9-10.

8. The in vitro rapid propagation method of Radix Polygoni Multiflori according to claim 1, characterized in that: In step (4), the rooting medium is 1 / 2MS rooting medium, the rooting culture temperature is 25±2°C, the humidity is 100%, the light intensity is 3000-5000Lux, the light time is 12-14 hours, and the culture time is 25-35 days.

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