A tissue culture seedling raising method of diyang-jiang-ting

Abstract

The application discloses a tissue culture seedling raising method of Yunnan polygonatum sibiricum, and belongs to the technical field of biology, and comprises the following steps: S1, treatment of explants: selecting clean Yunnan polygonatum sibiricum tubers with buds, and culturing the tubers under high-temperature stress at 40-45 DEG C for 12-24 h, and then directly transferring the tubers to low-temperature stress treatment at 2-4 DEG C for 24-36 h; S2, induction of callus: taking the treated tuber buds as explants, inoculating the explants into a callus induction culture medium after disinfection, and dark culture for 10-12 days; the callus induction culture medium comprises 1 / 2MS+2,4-D 1-2 mg / L, 6-BA 2-3 mg / L, NAA 0.3-0.6 mg / L and 1wt%-3wt% cactus juice; S3, induction of bud differentiation and induction of rooting. The tissue culture seedling raising method of Yunnan polygonatum sibiricum can effectively reduce the browning rate in induction of callus of Yunnan polygonatum sibiricum with tuber buds as explants.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to a method for tissue culture seedlings of Polygonatum yunnanense. Background Technology

[0002] Yunnan Polygonatum Pohygonatumkingianum Liliaceae Liliaceae Polygonatum Pobygonatum It is a perennial herbaceous medicinal plant with rhizomes, mainly distributed in Guangxi, Sichuan, Guizhou and Yunnan. It was first recorded in "Mingyi Bielu". Its rhizome is neutral in nature and sweet in taste. It has the effects of replenishing qi and nourishing yin, strengthening the spleen, moistening the lungs and benefiting the kidneys. It is used for symptoms of yin deficiency and lung dryness, yin and blood deficiency and spleen and stomach weakness.

[0003] Currently, medicinal Polygonatum yunnanense is mainly obtained through wild harvesting and artificial cultivation. Due to the limited wild resources, most medicinal Polygonatum yunnanense is artificially cultivated. At present, plant tissue culture technology is often used to quickly propagate Polygonatum yunnanense seedlings. However, browning of callus tissue is prone to occur during the tissue culture propagation of Polygonatum yunnanense, which affects the propagation efficiency of tissue culture seedlings. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, the present invention proposes a method for tissue culture seedlings of Polygonatum yunnanense.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0006] A method for tissue culture of Polygonatum yunnanense seedlings includes the following steps:

[0007] S1, Treatment of explants: Select cleaned rhizomes of Polygonatum yunnanensis with buds and culture them under high temperature stress at 40-45℃ for 12-24h, and then directly transfer them to low temperature stress at 2-4℃ for 24-36h.

[0008] S2, Callus induction: Take the tuber buds treated in step S1 as explants, disinfect the explants, and inoculate them into callus induction medium. Incubate in the dark for 10-12 days. The callus induction medium contains: 1 / 2 MS + 2,4-D 1-2 mg / L, 6-BA 2-3 mg / L, NAA 0.3-0.6 mg / L, and 1 wt%-3 wt% cactus juice.

[0009] S3, induces bud differentiation and rooting.

[0010] Furthermore, the method for inducing shoot differentiation is as follows: the swollen callus tissue is cut into small pieces and inoculated into a shoot induction medium. The shoot induction medium consists of MS + 6-BA 1.0-3.0 mg / L, 2,4-D 0.2-0.4 mg / L, LTDZ 0.1-0.2 mg / L and sucrose 35 g / L, and cultured for 25-30 days under a light intensity of 1500-2000 lux.

[0011] Furthermore, the method for inducing rooting is as follows: separate clustered buds to obtain single buds, inoculate the single buds into rooting medium, the rooting medium being: 1 / 2 MS + IBA 0.8-1.5 mg / L, the light intensity for rooting culture being 8000 lux-9000 lux, and the light exposure being 12-16 hours per day.

[0012] Furthermore, the disinfection method for the explants is as follows: after soaking the explants in 75% ethanol for 30 seconds, they are transferred to 5% sodium hypochlorite solution for 15 minutes, then disinfected with 0.1% mercuric chloride for 8 minutes, and finally rinsed with sterile water 3-4 times.

[0013] Through the above technical solution, the present invention can achieve at least the following beneficial effects: the method for tissue culture of Polygonatum yunnanense described in the present invention can effectively reduce the browning rate in the induction of callus tissue of Polygonatum yunnanense using tuber buds as explants. Detailed Implementation

[0014] Unless otherwise stated, all materials and reagents used in this invention are commercially available.

[0015] Example 1

[0016] A method for tissue culture of Polygonatum yunnanense seedlings includes the following steps:

[0017] S1, Treatment of explants: Select cleaned rhizomes of Polygonatum yunnanensis with buds and culture them under high temperature stress at 43℃ for 18h, and then directly transfer them to low temperature stress at 3℃ for 30h.

[0018] S2, Callus induction: Take the tuber buds treated in step S1 as explants, disinfect the explants, and inoculate them into callus induction medium and culture them in the dark for 11 days; The callus induction medium contains: 1 / 2MS + 2,4-D 1.5mg / L, 6-BA 2.5mg / L, NAA 0.5mg / L, and 2wt% cactus juice; The cactus juice is prepared by purchasing commercially available fresh cactus, juicing it with a juicer, passing it through 400-mesh gauze, and collecting the filtrate.

[0019] S3, induces bud differentiation and rooting.

[0020] The method for inducing shoot differentiation was as follows: the enlarged callus tissue was cut into small pieces and inoculated into the shoot induction medium, which consisted of MS + 6-BA 2 mg / L, 2,4-D 0.3 mg / L, LTDZ 0.15 mg / L and sucrose 35 g / L, and cultured for 28 days under a light intensity of 2000 lux.

[0021] The method for inducing rooting is as follows: separate clustered buds to obtain single buds, inoculate the single buds into rooting medium, the rooting medium is 1 / 2 MS + IBA 1.1 mg / L, the light intensity of the rooting culture is 8000 lux, and the light is 14 hours per day.

[0022] The disinfection method for explants is as follows: after soaking the explants in 75% ethanol for 30 seconds, they are transferred to 5% sodium hypochlorite solution for 15 minutes, then disinfected with 0.1% mercuric chloride for 8 minutes, and finally rinsed with sterile water 4 times.

[0023] Example 2

[0024] A method for tissue culture of Polygonatum yunnanense seedlings includes the following steps:

[0025] S1, Treatment of explants: Select cleaned rhizomes of Polygonatum yunnanensis with buds and culture them under high temperature stress at 40℃ for 12h, and then directly transfer them to low temperature stress at 2℃ for 24h.

[0026] S2, Inducing callus: Take the tuber buds treated in step S1 as explants, disinfect the explants, and inoculate them into callus induction medium and culture them in the dark for 10 days; The callus induction medium contains: 1 / 2MS + 2,4-D 1mg / L, 6-BA 2mg / L, NAA 0.3mg / L, and 1wt% cactus juice; The cactus juice is prepared by purchasing commercially available fresh cactus, juicing it with a juicer, passing it through 400-mesh gauze, and collecting the filtrate.

[0027] S3, induces bud differentiation and rooting.

[0028] The method for inducing shoot differentiation was as follows: the enlarged callus tissue was cut into small pieces and inoculated into the shoot induction medium, which consisted of MS + 1.0 mg / L 6-BA, 0.2 mg / L 2,4-D, 0.1 mg / L LTDZ and 35 g / L sucrose, and cultured for 25 days under a light intensity of 1500 lux.

[0029] The method for inducing rooting is as follows: separate clustered buds to obtain single buds, inoculate the single buds into rooting medium, the rooting medium is 1 / 2 MS + IBA 0.8 mg / L, the light intensity of the rooting culture is 8000 lux, and the light is 12 hours per day.

[0030] The disinfection method for explants is as follows: after soaking the explants in 75% ethanol for 30 seconds, they are transferred to 5% sodium hypochlorite solution for 15 minutes, then disinfected with 0.1% mercuric chloride for 8 minutes, and finally rinsed three times with sterile water.

[0031] Example 3

[0032] A method for tissue culture of Polygonatum yunnanense seedlings includes the following steps:

[0033] S1, Treatment of explants: Washed tubers of Polygonatum yunnanense with buds were cultured under high temperature stress at 45℃ for 24h, and then directly transferred to low temperature stress at 4℃ for 36h.

[0034] S2, Induction of callus: Take the tuber buds treated in step S1 as explants, disinfect the explants and inoculate them into callus induction medium, and culture them in the dark for 12 days; The callus induction medium contains: 1 / 2MS + 2,4-D 2mg / L, 6-BA 3mg / L, NAA 0.6mg / L, and 3wt% cactus juice; The cactus juice is prepared by purchasing commercially available fresh cactus, juicing it with a juicer, passing it through 400-mesh gauze and collecting the filtrate.

[0035] S3, induces bud differentiation and rooting.

[0036] The method for inducing shoot differentiation was as follows: the enlarged callus tissue was cut into small pieces and inoculated into the shoot induction medium, which consisted of MS + 6-BA 3.0 mg / L, 2,4-D 0.4 mg / L, LTDZ 0.2 mg / L and sucrose 35 g / L, and cultured for 30 days under a light intensity of 2000 lux.

[0037] The method for inducing rooting is as follows: separate clustered buds to obtain single buds, inoculate the single buds into rooting medium, the rooting medium is 1 / 2 MS + IBA 1.5 mg / L, the light intensity of the rooting culture is 9000 lux, and the light is 16 hours per day.

[0038] The disinfection method for explants is as follows: after soaking the explants in 75% ethanol for 30 seconds, they are transferred to 5% sodium hypochlorite solution for 15 minutes, then disinfected with 0.1% mercuric chloride for 8 minutes, and finally rinsed with sterile water 4 times.

[0039] Comparative Example 1

[0040] The difference between Comparative Example 1 and Example 1 is that the explants were not subjected to high-temperature stress treatment.

[0041] Comparative Example 2

[0042] The difference between Comparative Example 2 and Example 1 is that the explants were not subjected to low-temperature stress treatment.

[0043] Comparative Example 3

[0044] The difference between Comparative Example 3 and Example 1 is that the explants were neither subjected to high-temperature stress nor low-temperature stress treatment.

[0045] Experimental Example

[0046] Following the technical solutions of Examples 1 to 3 and Comparative Examples 1 to 3, the callus tissue was propagated and the browning rate was statistically analyzed. The results are shown in Table 1 below.

[0047] The browning rate of callus tissue is calculated as follows: (Number of browned explants / Number of inoculated explants) * 100%

[0048] Table 1. Statistical table of browning rate of callus under different treatments

[0049]

[0050] As can be seen from Table 1 above, the method for tissue culture of Polygonatum yunnanensis of the present invention can effectively reduce the browning rate of callus tissue and improve the efficiency of tissue culture seedlings.

[0051] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A tissue culture seedling raising method of Polygonatum franchetii, characterized by comprising the following steps: It includes the following steps: ​ S1, Treatment of explants: Select cleaned rhizomes of Polygonatum yunnanensis with buds and treat them with high temperature stress at 40-45℃ for 12-24h, and then directly transfer them to low temperature stress at 2-4℃ for 24-36h. S2, Callus induction: Take the tuber buds treated in step S1 as explants, disinfect the explants, and inoculate them into callus induction medium. Culture in the dark for 10-12 days. The callus induction medium is: 1 / 2 MS + 2,4-D 1-2 mg / L + 6-BA 2-3 mg / L + NAA 0.3-0.6 mg / L + 1wt%-3wt% cactus juice. S3, inducing shoot differentiation and inducing rooting; the method for inducing shoot differentiation is as follows: the swollen callus tissue is cut into small pieces and inoculated into the shoot induction medium, which is: MS + 6-BA 1.0-3.0 mg / L + 2,4-D 0.2-0.4 mg / L + TDZ 0.1-0.2 mg / L + sucrose 35 g / L, and cultured for 25-30 days under a light intensity of 1500-2000 lux; the method for inducing rooting is as follows: single shoots are obtained by separating clustered shoots, and the single shoots are inoculated into the rooting medium, which is: 1 / 2 MS + IBA 0.8-1.5 mg / L, and the rooting culture is carried out under a light intensity of 8000-9000 lux for 12-16 hours per day.

2. The tissue culture seedling raising method of Polygonatum franchetii according to claim 1, characterized in that: The disinfection method for the explants is as follows: after soaking the explants in 75% ethanol for 30 seconds, they are transferred to 5% sodium hypochlorite solution for 15 minutes, then disinfected with 0.1% mercuric chloride for 8 minutes, and finally rinsed with sterile water 3-4 times.

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