Stellaria yunnanensis tissue rapid propagation method and application

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a stellaria yunnanensis tissue rapid propagation method and application thereof.The stellaria yunnanensis tissue rapid propagation method comprises the steps that newly-germinated twigs are cut from a stellaria yunnanensis plant, cleaned and sterilized, and sterilized explants are inoculated into an induction culture medium according to the plant growth polarity to be cultured for 15-25 d; cutting the induced cluster buds into single buds, inoculating the single buds into a proliferation culture medium, culturing for 25-35 days, cutting off leaves after the cluster buds are cut into single buds, inoculating the single buds into a rooting culture medium, culturing for 20-30 days, transplanting seedlings subjected to rooting culture into a mixed matrix in a hole tray, culturing for 40-60 days, and transplanting. According to the method, the disinfection pollution rate of the explants can be controlled within 18%, and the disinfection death rate is controlled within 15%; the explant induction rate reaches 85% or above, the multiplication coefficient reaches 11.6 or above, the rooting rate reaches 98% or above, the survival rate after transplanting reaches 90% or above, and transplanting can be conducted after the culture period is only 100-150 days.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and particularly relates to a method and application for rapid tissue propagation of Stellaria yunnanensis Franch. Background Art

[0002] Stellaria yunnanensis Franch., also known as Yunnan chickweed, wheat ginseng, and bone grass, is a perennial herbaceous plant of the genus Stellaria in the Caryophyllaceae family. Its roots are used as medicine, with the effects of tonifying qi and nourishing blood, strengthening the spleen and kidney, etc.; it is used for symptoms such as qi and blood weakness, listlessness, dizziness and palpitation, soreness and weakness of the waist and knees, irregular menstruation, etc.; it is an important medicine for treating women's deficiency, and is also a commonly used medicine in ethnic minorities and folk. Modern traditional Chinese medicine research shows that the main components of Stellaria yunnanensis Franch. include various chemical components, such as flavonoids, phenolic acids, triterpenoids, etc. Among them, flavonoid compounds, such as quercetin, kaempferol, etc., have various biological activities such as antioxidant, anti-inflammatory, and antibacterial; while phenolic acid compounds, such as caffeic acid, chlorogenic acid, etc., these components help regulate metabolism in the body and protect the liver. It is a good medicinal resource with high development and utilization value.

[0003] Currently, there are very few medicinal plant resources of Stellaria yunnanensis Franch. In addition, due to over-excavation and its limited self-reproductive ability, the wild resources of Stellaria yunnanensis Franch. are becoming increasingly scarce. The traditional methods of seed propagation and ramet propagation not only have a low propagation coefficient, but are also greatly affected by seasonal and environmental factors, making it difficult to meet the growing market demand and resource protection requirements for Stellaria yunnanensis Franch. There is little research on the biotechnology of artificial propagation of Stellaria yunnanensis Franch. plants, and there have been no research reports on tissue culture in recent years. The publication number is: CN104996157A, and the name is: Cultivation method of wild Stellaria yunnanensis Franch. and food made therefrom, mainly through collecting the seeds of Stellaria yunnanensis Franch. for propagation; Sun Shiyao et al. (Sun Shiyao, Guo Ziwei, Zhang Bolin, et al. Response of seedling emergence and growth of Stellaria yunnanensis Franch. to soil moisture content [J]. Agricultural Science & Technology and Information, 2021(000 - 024). DOI:10.3969 / j.issn.1003 - 6997.2021.24.018.) studied the response of seed germination and growth of Stellaria yunnanensis Franch. to soil moisture content; Cao Zilin et al. (Cao Zilin, Wang Chunhua, Wang Xiaoli, et al. Response of seedling growth of Stellaria yunnanensis Franch. to light transmittance [J]. Agricultural Science & Technology and Information, 2022(003):000. DOI:10.3969 / j.issn.1003 - 6997.2022.03.007.) studied the response of seedling growth of Stellaria yunnanensis Franch. to light transmittance.

[0004] However, there are no research reports on tissue culture technology. At present, the tissue rapid propagation technology for Stellaria yunnanensis Franch. has not been perfected, and there is an urgent need for an efficient and stable tissue rapid propagation method for Stellaria yunnanensis Franch. Summary of the Invention

[0005] The object of the present invention is to provide a method and application for rapid tissue propagation of Stellaria yunnanensis Franch. in view of the problems existing in the prior art, so as to achieve the rapid propagation of Stellaria yunnanensis Franch.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for rapid tissue propagation of Stellaria yunnanensis Franch., the steps include:

[0007] 1) Selection and pretreatment of explants: Cut new germinated tender branches with a length of 15 - 20 cm from the Stellaria yunnanensis Franch. plant, cut off the leaves, rinse with tap water containing 1% (by volume) of dishwashing liquid for 10 - 30 min, and then rinse with tap water for 20 - 40 min;

[0008] 2) Sterilization of explants: Cut the pretreated tender branches into stem segments with a length of 1 - 2 cm with apical buds or axillary buds. Surface disinfect the explants with 75% alcohol for 20 - 30 s, rinse with sterile water 3 times, then soak and sterilize with 0.1% mercuric chloride solution for 8 - 12 min with shaking, and rinse with sterile water 3 - 5 times;

[0009] 3) Induction culture: Inoculate the sterilized explants in the induction medium according to the plant growth polarity and culture for 15 - 25 d;

[0010] The formula of the induction medium is: MS + cytokinin 0.5 - 4.0 mg / L + auxin 0.01 - 0.5 mg / L + sucrose or white sugar 30 g / L;

[0011] 4) Proliferation culture: Cut the induced cluster buds into single buds and inoculate them in the proliferation medium for culture for 25 - 35 d;

[0012] The formula of the proliferation medium is: MS + cytokinin 0.1 - 3.0 mg / L + auxin 0.01 - 0.5 mg / L + sucrose or white sugar 30 g / L;

[0013] 5) Rooting culture: Cut the cluster buds obtained by proliferation culture into single buds, cut off the leaves, and inoculate them in the rooting medium for culture for 20 - 30 d;

[0014] The formula of the rooting medium is: 1 / 2MS + auxin 0.1 - 2.0 mg / L + activated carbon 0.1 - 0.5 g / L + sucrose or white sugar 20 g / L;

[0015] 6) Acclimatization and transplantation: Transplant the seedlings obtained by rooting culture into the mixed substrate in a plug tray, and transplant them after cultivating for 40 - 60 d;

[0016] The cytokinin is BA or TDZ, and the auxin is IBA or NAA.

[0017] Further, in steps (3), (4) and (5), the culture conditions are as follows: the temperature is 25 ± 2 °C, the light intensity of illumination is 2000 - 3000 Lx, and the illumination time is 10 - 14 h / d.

[0018] Further, in step (3), the formula of the induction medium is: MS + cytokinin 2.0 mg / L + auxin 0.1 mg / L + sucrose or white sugar 30 g / L.

[0019] Further, in step (4), the formula of the proliferation medium is: MS + cytokinin 1.0 mg / L + auxin 0.05 mg / L + sucrose or white sugar 30 g / L.

[0020] Further, in step (5), the formula of the rooting medium is: 1 / 2 MS + auxin 2.0 mg / L + activated carbon 0.5 g / L + sucrose or white sugar 20 g / L.

[0021] Further, in step (6), the mixed substrate is prepared from peat soil, perlite, humus soil, and coarse sand in a volume ratio of 4:2:2:1.

[0022] The beneficial technical effects of the present invention are as follows: The present invention provides a method for rapid tissue propagation of Silene acaulis var. lanceolata. Using 75% alcohol for surface disinfection and then combining with soaking and shaking sterilization in 0.1% mercuric chloride solution, the disinfection contamination rate of the explant can be controlled within 18%, and the disinfection mortality rate can be controlled within 15%. Selecting MS as the basic medium and combining with appropriate concentrations of cytokinin (BA or TDZ) and growth hormone (IBA or NAA), the induction rate of the explant reaches more than 85%, the proliferation coefficient reaches more than 11.6, the rooting rate is as high as more than 98%, the survival rate after transplantation reaches more than 90%, and the culture period is only 100 - 150 days before transplantation can be carried out. It solves the problems of the traditional propagation method such as long time consumption and being easily affected by seasonal and environmental factors; fills the gap in the tissue propagation technology of Silene acaulis var. lanceolata, realizes the rapid and large-scale propagation of Silene acaulis var. lanceolata, meets the market demand and protects wild resources. Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 The seedlings induced in Example 3 of the present invention for 20 days.

[0025] Figure 2The seedlings cultured by proliferation for 30 days in Example 3 of the present invention.

[0026] Figure 3 The seedlings cultured by rooting for 25 days in Example 3 of the present invention.

[0027] Figure 4 The seedlings acclimatized for 50 days in Example 3 of the present invention. Detailed implementation manners

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] Example 1

[0030] A method for rapid tissue propagation of Silene vulgaris (Moench) Garcke, the steps include:

[0031] 1) Selection and pretreatment of explants: Cut a newly germinated tender branch with a length of 15 cm from the Silene vulgaris (Moench) Garcke plant, cut off the leaves, rinse with tap water containing 1% (by volume) of dishwashing liquid for 10 min, and then rinse with tap water for 20 min.

[0032] 2) Sterilization of explants: Cut the pretreated tender branch into stem segments with a length of 1 cm with apical buds or axillary buds. The explants are surface sterilized with 75% alcohol for 20 s, rinsed 3 times with sterile water, then soaked and sterilized with 0.1% mercuric chloride solution for 8 min with shaking, and rinsed 5 times with sterile water.

[0033] 3) Induction culture: Inoculate the sterilized explants on the induction medium (formula: MS + BA 0.5 mg / L + IBA 0.01 mg / L + sucrose 30 g / L) according to the plant growth polarity, at a temperature of 25 ± 2 °C, a light intensity of 2000 Lx, and a light time of 10 h / d, and culture for 15 d.

[0034] 4) Proliferation culture: Cut the induced clustered buds into single buds and inoculate them into the proliferation medium (formula: MS + BA 0.1 mg / L + IBA 0.01 mg / L + sucrose 30 g / L), at a temperature of 25 ± 2 °C, a light intensity of 2000 Lx, and a light time of 10 h / d, and culture for 25 d.

[0035] 5) Rooting culture: Cut the clustered buds cultured by proliferation into single buds, cut off the leaves, and inoculate them into the rooting medium (formula: 1 / 2MS + IBA 0.1 mg / L + activated carbon 0.1 g / L + sucrose 20 g / L), at a temperature of 25 ± 2 °C, a light intensity of 2000 Lx, and a light time of 10 h / d, and culture for 20 d.

[0036] 6) Hardening and transplanting of seedlings: The rooted seedlings are transplanted into the mixed matrix in the plug tray (the mixed matrix is ​​peat soil, perlite, humus soil, and coarse sand in a volume ratio of 4: 2: 2: 1) and transplanted after 40 days of cultivation.

[0037] In actual operation, cytokinin can be replaced by TDZ, auxin can be replaced by NAA, and sucrose can be replaced by white sugar.

[0038] Example 2

[0039] A method for rapid propagation of spatholobiaceae tissue, comprising the following steps:

[0040] 1) Explant selection and pretreatment: Newly sprouted shoots 20 cm long were cut from the plants of Millefiori, and the leaves were cut off. The shoots were rinsed with tap water containing 1% by volume detergent for 30 minutes, and then rinsed with tap water for 40 minutes.

[0041] 2) Explant sterilization: The pretreated shoots were cut into 2 cm long stem segments with terminal buds or axillary buds. The explants were surface disinfected with 75% alcohol for 30 seconds, rinsed with sterile water three times, then immersed in 0.1% mercuric chloride solution for 12 minutes and rinsed with sterile water seven times.

[0042] 3) Induction culture: The sterilized explants were inoculated into the induction medium (formula: MS+BA4.0mg / L+IBA 0.5mg / L+sucrose 30g / L) according to the plant growth polarity, with the temperature at 25±2℃, light intensity at 3000Lx, light duration at 14h / d, and cultured for 25 days.

[0043] 4) Proliferation culture: The induced clustered buds were cut into single buds and inoculated into proliferation medium (formula: MS+BA 3.0mg / L+IBA 0.5mg / L+sucrose 30g / L), with a temperature of 25±2℃, a light intensity of 3000Lx, a light duration of 14h / d, and cultured for 35 days.

[0044] 5) Rooting culture: Cut the clustered buds into single buds and remove the leaves. Inoculate them into rooting medium (formula: 1 / 2 MS + IBA 1.0 mg / L + activated carbon 0.5 g / L + sucrose 20 g / L), with a temperature of 25±2℃, a light intensity of 3000Lx, and a light duration of 14h / d, and culture for 30 days.

[0045] 6) Hardening and transplanting of seedlings: The rooted seedlings are transplanted into the mixed matrix in the plug tray (the mixed matrix is ​​peat soil, perlite, humus soil, and coarse sand in a volume ratio of 4: 2: 2: 1) and transplanted after 60 days of cultivation.

[0046] In the actual operation process, cytokinin can be replaced by TDZ, auxin can be replaced by NAA, and sucrose can be replaced by white sugar.

[0047] Example 3

[0048] A method for rapid tissue propagation of Silene vulgaris var. lanceolata, the steps include:

[0049] 1) Selection and pretreatment of explants: Cut a newly germinated tender branch with a length of 17 cm from the Silene vulgaris var. lanceolata plant, cut off the leaves, rinse with tap water containing 1% (by volume) of dishwashing liquid for 20 min, and then rinse with tap water for 30 min.

[0050] 2) Sterilization of explants: Cut the pretreated tender branch into stem segments with a length of 1.5 cm with apical buds or axillary buds. The explants are surface-sterilized with 75% alcohol for 25 s, rinsed 3 times with sterile water, then soaked and sterilized with 0.1% mercuric chloride solution for 10 min with shaking, and rinsed 6 times with sterile water.

[0051] 3) Induction culture: Inoculate the sterilized explants on the induction medium (formula: MS + BA 2.0 mg / L + IBA 0.1 mg / L + sucrose or white sugar 30 g / L) according to the plant growth polarity, at a temperature of 25 ± 2 °C, a light intensity of 2500 Lx, and a light time of 12 h / d, and culture for 20 d. The results are as Figure 1 shown.

[0052] 4) Proliferation culture: Cut the induced clustered buds into single buds and inoculate them into the proliferation medium (formula: MS + BA 1.0 mg / L + IBA 0.05 mg / L + sucrose or white sugar 30 g / L), at a temperature of 25 ± 2 °C, a light intensity of 2500 Lx, and a light time of 12 h / d, and culture for 30 d. The results are as Figure 2 shown.

[0053] 5) Rooting culture: Cut the leaves off the clustered buds obtained from the proliferation culture and cut them into single buds, and inoculate them into the rooting medium (formula: 1 / 2 MS + IBA 2.0 mg / L + activated carbon 0.5 g / L + sucrose or white sugar 20 g / L), at a temperature of 25 ± 2 °C, a light intensity of 2500 Lx, and a light time of 12 h / d, and culture for 25 d. The results are as Figure 3 shown.

[0054] 6) Acclimatization and transplantation: Transplant the seedlings obtained from the rooting culture into the mixed substrate in the plug tray (the mixed substrate is prepared from peat soil, perlite, humus soil, and coarse sand in a volume ratio of 4:2:2:1), cultivate for 50 d and then transplant. The seedling raising results are as Figure 4 shown.

[0055] In the actual operation process, cytokinin can be replaced by TDZ, auxin can be replaced by NAA, and sucrose can be replaced by white sugar.

[0056] For Examples 1 - 3, the disinfection mortality rate, disinfection contamination rate, primary induction rate, proliferation coefficient, rooting rate, and transplanting survival rate were statistically analyzed, and the results are shown in Table 1 below.

[0057] Table 1 Statistical Results

[0058] Disinfection mortality rate Disinfection contamination rate Primary induction rate Proliferation coefficient Rooting rate Transplanting survival rate Example 1 15% 18% 85% 11.6 98% 90% Example 2 13% 10% 92% 13.6 99% 93% Example 3 8% 11% 95% 16.8 100% 97%

[0059] As can be seen from Table 1, the disinfection contamination rate of the explants was controlled within 18%, and the disinfection mortality rate was controlled within 15%; the induction rate of the explants reached over 85%, the proliferation coefficient reached over 11.6, the rooting rate was as high as over 98%, and the survival rate after transplanting reached over 90%.

[0060] Finally, it should be noted that the above examples are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above examples, those of ordinary skill in the art should understand that: the present invention can still be modified or equivalently replaced, and any modification or partial replacement without departing from the spirit and scope of the present invention should be covered by the scope of the claims of the present invention.

Claims

1. A method for rapid propagation of Thousand Needle Grass tissue, characterized in that the steps include: 1) Explant selection and pretreatment: Cut newly sprouted shoots 15 to 20 cm long from the plants of the Thousand Needles Grass, cut off the leaves, rinse with tap water with 1% volume ratio of detergent for 10 to 30 minutes, and then rinse with tap water for 20 to 40 minutes; 2) Explant sterilization: The pretreated shoots were cut into 1-2 cm long stem segments with terminal buds or axillary buds. The explants were surface sterilized with 75% alcohol for 20-30 seconds, rinsed with sterile water three times, then immersed in 0.1% mercuric chloride solution for 8-12 minutes and rinsed with sterile water for 3-5 times. 3) Induction culture: sterilized explants are inoculated in the induction medium according to the plant growth polarity and cultured for 15 to 25 days; The formula of the induction medium is: MS+cytokinin 0.5-4.0 mg / L+auxin 0.01-0.5 mg / L+sucrose or white sugar 30 g / L; 4) Proliferation culture: The induced clustered buds are cut into single buds and inoculated into the proliferation medium for 25 to 35 days; The formula of the proliferation culture medium is: MS+cytokinin 0.1-3.0 mg / L+auxin 0.01-0.5 mg / L+sucrose or white sugar 30 g / L; 5) Rooting culture: Cut the clustered buds into single buds, remove the leaves, and inoculate them in rooting medium for 20 to 30 days; The formula of the rooting medium is: 1 / 2MS+auxin 0.1-2.0 mg / L+activated carbon 0.1-0.5 g / L+sucrose or white sugar 20 g / L; 6) Hardening and transplanting: The rooted seedlings are transplanted into the mixed medium in the plug tray and transplanted after 40 to 60 days of cultivation; The cytokinin is BA or TDZ, and the auxin is IBA or NAA.

2. The method according to claim 1, characterized in that: In steps (3), (4) and (5), the culture conditions are: temperature of 25±2°C, light intensity of 2000-3000Lx, and illumination time of 10-14h / d.

3. The method according to claim 1, characterized in that: In step (3), the formula of the induction medium is: MS+cytokinin 2.0 mg / L+auxin 0.1 mg / L+sucrose or white sugar 30 g / L.

4. The method according to claim 1, characterized in that: In step (4), the formula of the proliferation medium is: MS+cytokinin 1.0 mg / L+auxin 0.05 mg / L+sucrose or white sugar 30 g / L.

5. The method according to claim 1, characterized in that: In step (5), the formula of the rooting medium is: 1 / 2MS+2.0 mg / L auxin+0.5 g / L activated carbon+20 g / L sucrose or white sugar.

6. The method according to claim 1, characterized in that: In step (6), the mixed matrix is ​​prepared by mixing peat soil, perlite, humus soil and coarse sand in a volume ratio of 4:2:2:

1.

7. Use of the method according to any one of claims 1 to 6 in the rapid propagation of Thunbergia lappa tissue.

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