Tissue culture and rapid propagation culture medium composition and rapid propagation method of Leukospermum maritimum

Abstract

The invention discloses a tissue culture and rapid propagation culture medium composition and a rapid propagation method of marina gromwell. Relates to the technical field of tissue culture rapid propagation. Comprising one or more of a primary culture medium, a subculture medium and a rooting culture medium. According to the tissue culture and rapid propagation method of the Leukebia quinata, the plant tissue culture technology is applied, the scapes are adopted as explants, the materials are easy to obtain, and the screened subculture medium and rooting medium solve the problem of large-scale culture of the Leukebia quinata. Especially, in the rooting culture process, an improved 1 / 8 MS nutrient solution is directly selected to add auxin, the step of preparing a culture medium is omitted, the lithospermum maritimum can root, the rooting rate can reach 90%, the root culture medium does not need to be washed during transplanting, and operation is easy. According to the method, the tissue culture procedure is simplified, the production cost is saved, and the method is simple, convenient and feasible, and has important significance on large-scale seedling culture of the Leukemia maritima.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture rapid propagation, and more particularly to a tissue culture rapid propagation medium combination of large-leaf Lithospermum officinale and a rapid propagation method thereof. Background Art

[0002] Mertensia sibirica (L.) G.Don, belonging to the Boraginaceae family, is a perennial herb with slender or shortened rhizomes, basal leaves clustered, cauline leaves alternate, cymes, usually small panicles integrated on the upper part of the stem, without bracts, funnel-shaped corolla, usually blue or light blue, and tetrahedral nuts. Mertensia sibirica has a large annual growth, a large number of flowers, blue flowers, and seeds containing γ-linolenic acid (GLA) accounting for 22.8% of the fatty acid content, which has unique ornamental and economic value. In the wild, it is only naturally distributed in a narrow area on high-altitude slopes above 2,200 meters, and the population is sparse.

[0003] At present, there is no report on the breeding technology of large-leafed lithospermum. In order to rationally develop and utilize this potential plant, the plant tissue culture propagation method can meet the needs of rapid propagation of seedlings in a short period of time.

[0004] Therefore, whether it is possible to provide a tissue culture rapid propagation medium combination and a rapid propagation method for large-leaf Lithospermum officinale is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a tissue culture rapid propagation culture medium combination of large-leaf Lithospermum officinale and a rapid propagation method thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A tissue culture rapid propagation medium combination of large-leaf Lithospermum erythrorhizon, comprising a plurality of primary culture medium, secondary culture medium and rooting culture medium;

[0008] The primary culture medium included: MS, 1.5 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0009] The subculture medium includes: MS, 0.5-1.0 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0010] Rooting medium includes: 1 / 8MS, 0.1mg / L IBA.

[0011] Preferred: including primary culture medium, secondary culture medium, and rooting culture medium;

[0012] The primary culture medium includes: MS, 1.5 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0013] Subculture medium includes: MS, 0.8 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0014] Rooting medium includes: 1 / 8MS, 0.1mg / L IBA.

[0015] The present invention also provides a rapid propagation method using the above culture medium combination, comprising the following steps:

[0016] (1) Disinfection of scapes;

[0017] (2) Primary culture: remove the two ends of the sterilized scape, cut the scape into small segments along the cross section, inoculate the scape in the primary culture medium, and culture it to obtain callus tissue and adventitious buds;

[0018] (3) Subculture: the adventitious buds obtained in step (2) are cut and inoculated into a subculture proliferation medium for culture;

[0019] (4) Rooting culture: The adventitious buds obtained in step (3) are individually inoculated into sterile water / rooting medium / culture solution to obtain test tube plantlets;

[0020] (5) Hardening the seedlings: placing the test tube seedlings cultured in step (4) in a hardening room to harden the seedlings and prepare for transplanting;

[0021] (6) Transplantation.

[0022] Preferably, step (1) is specifically as follows: select young and tender scapes, soak them in washing powder for 10 minutes, rinse them with tap water, dry them with filter paper, rinse them with sterile water once, then disinfect them with 75% alcohol solution for 30 seconds, rinse them with sterile water twice, then disinfect them with 0.1% HgCl2 for 1 minute, rinse them with sterile water 5 times, dry them with sterile filter paper and set aside.

[0023] Preferably, step (2) is specifically as follows: using a sterile scalpel to partially remove the two ends of the disinfected scape, cutting the scape into small segments of 1.0 cm along the cross section, inoculating the scape in a primary culture medium and culturing for 35 to 50 days to obtain callus tissue and adventitious buds; the culture conditions are: 22 to 25° C., light intensity 2000 to 2500 Lux, and a photoperiod of 12 h / d.

[0024] Preferably, step (3) is specifically as follows: the adventitious buds in step (2) are cut and inoculated into a subculture proliferation medium, respectively, at 22-25° C., with a light intensity of 2000-2500 Lux and a photoperiod of 12 h / d, and cultured for 20-25 days.

[0025] Preferably, step (4) is specifically as follows: a single adventitious bud with a height of 5 cm obtained by culture in step (3) is cultured in sterile water at 22-25° C., a light intensity of 2000-2500 Lux, and a photoperiod of 12 h / d to obtain a test tube seedling;

[0026] or,

[0027] The adventitious buds cultured in step (3) with a height of 5 cm are individually inoculated into 1 / 8MS culture solution and cultured at 22-25° C., a light intensity of 2000-2500 Lux, and a photoperiod of 12 h / d to obtain test tube seedlings;

[0028] or,

[0029] The adventitious buds cultured in step (3) with a height of 5 cm are individually inoculated into a rooting medium and cultured at 22-25° C., a light intensity of 2000-2500 Lux, and a photoperiod of 12 h / d to obtain test tube seedlings.

[0030] Preferably, step (5) specifically comprises: when the roots of the test tube seedlings cultured in step (4) grow to 1-2 cm, hardening the seedlings for 3 days to obtain seedlings for transplanting.

[0031] Preferably, step (6) is specifically as follows: taking out the seedlings cultured in step (5), transplanting them into a test tube seedling transplanting medium, placing them into a small arch shed in a greenhouse, maintaining the humidity of the arch shed at 80% to 85% and the temperature at 20 to 25° C., gradually opening the small arch shed one week after transplanting, and entering routine management after 30 days;

[0032] The test tube seedling transplanting matrix is ​​peat soil, perlite and vermiculite in a mass ratio of 3:1:1. Before transplanting, the matrix is ​​disinfected with 1000 times diluted 50% carbendazim wettable powder.

[0033] The present invention also provides the application of the above culture medium combination or any one of the above rapid propagation methods in agricultural production.

[0034] It can be known from the above technical scheme that compared with the prior art, the present invention discloses a tissue culture rapid propagation culture medium combination and a rapid propagation method of large-leafed purpurogenous grass, and the technical effect obtained is the tissue culture rapid propagation method of large-leafed purpurogenous grass provided by the present invention, by using plant tissue culture technology, using scape as explant, easy to obtain materials, and the screened subculture culture medium and rooting culture medium solve the problem of large-leafed purpurogenous grass being cultured on a large scale. Especially in the rooting culture process, directly selecting the improved 1 / 8MS nutrient solution to add auxin, omitting the step of preparing culture medium, large-leafed purpurogenous grass can also be rooted, and the rooting rate can reach 90%, and the root culture medium does not need to be washed during transplanting, and the operation is simple. This method simplifies the tissue culture procedure, saves production costs, is simple and easy to operate, and has important significance for the large-scale seedling cultivation of large-leafed purpurogenous grass. DETAILED DESCRIPTION

[0035] The following will be described clearly and completely in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The embodiment of the invention discloses a tissue culture rapid propagation culture medium combination of large-leaf Lithospermum officinale and a rapid propagation method thereof.

[0037] Example 1

[0038] A tissue culture rapid propagation medium combination of large-leaf Lithospermum erythrorhizon, comprising a primary culture medium and a secondary culture medium;

[0039] The primary culture medium included: MS, 1.5 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0040] Subculture medium includes: MS, 0.5 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0041] The rapid propagation method comprises the following steps:

[0042] (1) Disinfection of flower stalks: Select young and tender flower stalks, soak them in washing powder for 10 min, rinse them with tap water, dry them with filter paper, place them in a sterile bottle, rinse them once with sterile water in a clean bench, disinfect them with 75% alcohol solution for 30 s, rinse them twice with sterile water, disinfect them with 0.1% HgCl2 for 1 min, rinse them 5 times with sterile water, dry them with sterile filter paper and set aside;

[0043] (2) Primary culture: Use a sterile scalpel to cut off the two ends of the disinfected scape, cut the scape into small segments of 1.0 cm along the cross section, and inoculate the scape in a medium containing MS + 1.5 mg / L 6-BA + 0.01 mg / L NAA + 30 g / L sucrose + 6 g / L agar, pH 5.8-6.2, and culture for 35-50 days to obtain callus tissue and adventitious buds;

[0044] The culture conditions were: 22-25°C, light intensity 2000-2500 Lux, photoperiod 12 h / d;

[0045] (3) Subculture: The adventitious buds in step (2) were cut and inoculated into subculture proliferation medium respectively, with MS medium as the basic medium, 0.5 mg / L 6-benzylaminoadenine (6-BA), 0.01 mg / L naphthaleneacetic acid (NAA), 30 g / L sucrose, and 6 g / L agar added to prepare a subculture medium with a pH of 5.8 to 6.2, and the culture conditions were the same as those in step (2), and the culture was carried out for 20 to 25 days (the results showed that the average proliferation coefficient was 3.14);

[0046] (4) Rooting culture: The adventitious buds with a height of 5 cm obtained in step (3) are individually inoculated into sterile water for culture under the same culture conditions as step (2). After 20 days, the rooting rate can reach 20%, and test tube seedlings are obtained;

[0047] (5) Hardening the seedlings: When the roots of the test tube seedlings cultured in step (4) grow to 1-2 cm, place them in a hardening room for 3 days to obtain seedlings, ready for transplanting;

[0048] (6) Transplantation: The test tube seedling transplanting matrix is ​​peat soil: perlite: vermiculite = 3:1:1. Before transplanting, the matrix is ​​disinfected with a 1000-fold dilution of 50% carbendazim wettable powder. The seedlings cultured in step (5) are taken out and transplanted into the test tube seedling transplanting matrix, and placed in a small arch shed in the greenhouse. The humidity of the arch shed is maintained at 80% to 85% and the temperature is maintained at 20 to 25°C. The small arch shed is gradually opened one week after transplanting, and routine management is carried out after 30 days.

[0049] Example 2

[0050] A tissue culture rapid propagation medium combination of large-leaf Lithospermum erythrorhizon, comprising one or more of a primary culture medium, a secondary culture medium, and a rooting culture medium;

[0051] The primary culture medium includes: MS, 1.5 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0052] Subculture medium includes: MS, 0.8 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0053] Rooting medium includes: 1 / 8MS, 0.1mg / L IBA.

[0054] The rapid propagation method comprises the following steps:

[0055] (1) Disinfection of flower stalks: Select young and tender flower stalks, soak them in washing powder for 10 min, rinse them with tap water, dry them with filter paper, place them in a sterile bottle, rinse them once with sterile water in a clean bench, disinfect them with 75% alcohol solution for 30 s, rinse them twice with sterile water, disinfect them with 0.1% HgCl2 for 1 min, rinse them 5 times with sterile water, dry them with sterile filter paper and set aside;

[0056] (2) Primary culture: Use a sterile scalpel to cut off the two ends of the disinfected scape, cut the scape into small segments of 1.0 cm along the cross section, and inoculate the scape in a medium containing MS + 1.5 mg / L 6-BA + 0.01 mg / L NAA + 30 g / L sucrose + 6 g / L agar, pH 5.8-6.2, and culture for 35-50 days to obtain callus tissue and adventitious buds;

[0057] The culture conditions are: 22-25°C, light intensity 2000-2500 Lux, and photoperiod of 12 h / d.

[0058] (3) Subculture: The adventitious buds in step (2) were cut and inoculated into subculture proliferation medium respectively, with MS medium as the basic medium, 0.8 mg / L 6-benzylaminoadenine (6-BA), 0.01 mg / L naphthaleneacetic acid (NAA), 30 g / L sucrose, and 6 g / L agar added to prepare a subculture medium with a pH of 5.8 to 6.2, and the culture conditions were the same as those in step (2), and the culture was carried out for 20 to 25 days (the results showed that the average proliferation coefficient was 4.37);

[0059] (4) Rooting culture: The adventitious buds cultured in step (3) with a height of 5 cm are individually inoculated into a rooting medium containing 1 / 8MS + 0.1 mg / LIBA and cultured under the same culture conditions as in step (2). After 20 days, the rooting rate can reach 90%, and test tube plantlets are obtained;

[0060] (5) Hardening the seedlings: When the roots of the test tube seedlings cultured in step (4) grow to 1-2 cm, place them in a hardening room for 3 days to obtain seedlings, ready for transplanting;

[0061] (6) Transplantation: The test tube seedling transplanting matrix is ​​peat soil: perlite: vermiculite = 3:1:1. Before transplanting, the matrix is ​​disinfected with a 1000-fold solution of 50% carbendazim wettable powder. The seedlings cultured in step (5) are taken out and transplanted into the matrix, and placed in a small arch shed in the greenhouse. The humidity of the arch shed is maintained at 80% to 85% and the temperature is maintained at 20 to 25°C. The small arch shed is gradually opened one week after transplanting, and routine management is implemented after 30 days.

[0062] Example 3

[0063] A tissue culture rapid propagation medium combination of large-leaf Lithospermum erythrorhizon, comprising a primary culture medium and a secondary culture medium;

[0064] The primary culture medium included: MS, 1.5 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0065] Subculture medium includes: MS, 1.0 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0066] The rapid propagation method comprises the following steps:

[0067] (1) Disinfection of flower stalks: Select young and tender flower stalks, soak them in detergent for 10 min, rinse them with tap water, dry them with filter paper, place them in a sterile bottle, rinse them once with sterile water in a clean bench, disinfect them with 75% alcohol solution for 30 s, rinse them twice with sterile water, disinfect them with 0.1% HgCl2 for 1 min, rinse them five times with sterile water, dry them with sterile filter paper and set aside.

[0068] (2) Primary culture: Use a sterile scalpel to cut off the two ends of the disinfected scape, cut the scape into small segments of 1.0 cm along the cross section, and inoculate the scape in a medium containing MS + 1.5 mg / L 6-BA + 0.01 mg / L NAA + 30 g / L sucrose + 6 g / L agar, pH 5.8-6.2, and culture for 35-50 days to obtain callus tissue and adventitious buds;

[0069] The culture conditions were: 22-25°C, light intensity 2000-2500 Lux, photoperiod 12 h / d;

[0070] (3) Subculture: The adventitious buds in step (2) were cut and inoculated into subculture proliferation medium respectively, using MS medium as the basic medium, adding 1.0 mg / L 6-benzylaminoadenine (6-BA), 0.01 mg / L naphthaleneacetic acid (NAA), 30 g / L sucrose, and 6 g / L agar to prepare a subculture medium with a pH of 5.8 to 6.2, and the culture conditions were the same as those in step (2), and the culture was carried out for 20 to 25 days (the results showed that the average proliferation coefficient was 4.15);

[0071] (4) Rooting culture: The adventitious buds cultured in step (3) with a height of 5 cm are individually inoculated into 1 / 8 MS culture solution and cultured under the same culture conditions as in step (2). After 20 days, the rooting rate can reach 75%, and test tube seedlings are obtained;

[0072] (5) Hardening the seedlings: When the roots of the test tube seedlings cultured in step (4) grow to 1 to 2 cm, place them in a hardening room for 3 days and prepare for transplanting;

[0073] (6) Transplantation: The test tube seedling transplanting matrix is ​​peat soil: perlite: vermiculite = 3:1:1. Before transplanting, the matrix is ​​disinfected with a 1000-fold solution of 50% carbendazim wettable powder. The seedlings cultured in step (5) are taken out and transplanted into the matrix, and placed in a small arch shed in the greenhouse. The humidity of the arch shed is maintained at 80% to 85% and the temperature is maintained at 20 to 25°C. The small arch shed is gradually opened one week after transplanting, and routine management is implemented after 30 days.

[0074] In summary, the optimal formula of subculture culture medium is: MS+0.8mg / L 6-BA+0.01mg / L NAA+30g / L sucrose+6g / L agar; 1 / 8MS+0.1mg / L IBA is used for rooting culture.

[0075] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0076] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A tissue culture rapid propagation medium combination for large-leaf Lithospermum officinale, characterized in that: including multiple of primary culture medium, subculture culture medium, and rooting culture medium; The primary culture medium comprises: MS, 1.5 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar; The subculture medium comprises: MS, 0.5-1.0 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar; The rooting medium comprises: 1 / 8MS and 0.1 mg / L IBA.

2. The culture medium combination according to claim 1, characterized in that Including primary culture medium, subculture culture medium, and rooting culture medium; The primary culture medium comprises: MS, 1.5 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar; The subculture medium comprises: MS, 0.8 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar; The rooting medium comprises: 1 / 8MS and 0.1 mg / L IBA.

3. A rapid propagation method using the culture medium combination according to claim 1 or 2, characterized in that: The following steps are involved: (1) Disinfection of scapes; (2) Primary culture: remove the two ends of the sterilized scape, cut the scape into small segments along the cross section, inoculate the scape in the primary culture medium, and culture it to obtain callus tissue and adventitious buds; (3) Subculture: the adventitious buds obtained in step (2) are cut and inoculated into a subculture proliferation medium for culture; (4) Rooting culture: The adventitious buds obtained in step (3) are individually inoculated into sterile water / rooting medium / culture solution to obtain test tube plantlets; (5) Hardening the seedlings: placing the test tube seedlings cultured in step (4) in a hardening room to harden the seedlings and prepare for transplanting; (6) Transplantation.

4. The rapid propagation method according to claim 3, characterized in that: Step (1) is specifically as follows: select young and tender scapes, soak them in washing powder for 10 minutes, rinse them with tap water, dry them with filter paper, rinse them with sterile water once, disinfect them with 75% alcohol solution for 30 seconds, rinse them with sterile water twice, disinfect them with 0.1% HgCl2 for 1 minute, rinse them with sterile water 5 times, dry them with sterile filter paper and set aside.

5. The rapid propagation method according to claim 4, characterized in that Step (2) is specifically as follows: using a sterile scalpel to partially remove the two ends of the disinfected scape, cutting the scape into small segments of 1.0 cm along the cross section, inoculating the scape in a primary culture medium and culturing for 35 to 50 days to obtain callus tissue and adventitious buds; the culture conditions are: 22 to 25° C., light intensity 2000 to 2500 Lux, and a photoperiod of 12 h / d.

6. The rapid propagation method according to claim 5, characterized in that: Step (3) is specifically as follows: the adventitious buds in step (2) are cut and inoculated into a subculture proliferation medium, respectively, at 22-25° C., with a light intensity of 2000-2500 Lux and a photoperiod of 12 h / d, for 20-25 days.

7. The rapid propagation method according to claim 6, characterized in that: Step (4) is specifically as follows: a single adventitious bud with a height of 5 cm obtained by culture in step (3) is placed in sterile water for culture; 22-25℃, light intensity 2000-2500Lux, photoperiod 12h / d, test tube seedlings were obtained; or, The adventitious buds cultured in step (3) with a height of 5 cm are individually inoculated into 1 / 8MS culture solution and cultured at 22-25° C., a light intensity of 2000-2500 Lux, and a photoperiod of 12 h / d to obtain test tube seedlings; or, The adventitious buds cultured in step (3) with a height of 5 cm are individually inoculated into a rooting medium and cultured at 22-25° C., a light intensity of 2000-2500 Lux, and a photoperiod of 12 h / d to obtain test tube seedlings.

8. The rapid propagation method according to claim 7, characterized in that: Step (5) is specifically as follows: when the roots of the test tube seedlings cultured in step (4) grow to 1-2 cm, harden the seedlings for 3 days to obtain seedlings, and prepare for transplanting.

9. The rapid propagation method according to claim 8, characterized in that: Step (6) specifically comprises: taking out the seedlings cultured in step (5), transplanting them into a test tube seedling transplanting medium, placing them into a small arch shed in a greenhouse, maintaining the humidity of the arch shed at 80% to 85% and the temperature at 20 to 25° C., gradually opening the small arch shed one week after transplanting, and entering routine management after 30 days; The test tube seedling transplanting matrix is ​​peat soil, perlite and vermiculite in a mass ratio of 3:1:1, and the matrix is ​​disinfected with 1000 times liquid of 50% carbendazim wettable powder before transplanting.

10. Use of the culture medium combination according to claim 1 or 2 or the rapid propagation method according to any one of claims 3 to 9 in agricultural production.