A tissue culture medium composition for rapid propagation of Lithospermum macrocephala and its rapid propagation method

Abstract

This invention discloses a rapid propagation culture medium combination and method for *Lithospermum macrocephalum* via tissue culture. It relates to the field of rapid tissue culture propagation technology. The medium includes one or more of a primary culture medium, a subculture medium, and a rooting medium, and provides a rapid propagation method. The rapid propagation method for *Lithospermum macrocephalum* via tissue culture provided by this invention utilizes plant tissue culture technology, using flower stalks as explants, which are readily available. The selected subculture and rooting media solve the problem of large-scale cultivation of *Lithospermum macrocephalum*. In particular, during the rooting culture process, a modified 1 / 8MS nutrient solution with added auxin is directly selected, eliminating the need for preparing the culture medium, and rooting of *Lithospermum macrocephalum* can still occur with a rooting rate of up to 90%. There is no need to wash the roots with the culture medium during transplanting, making the operation simple. This method simplifies the tissue culture procedure, saves production costs, and is easy to implement, which is of great significance for the large-scale seedling production of *Lithospermum macrocephalum*.

Description

Technical Field

[0001] This invention relates to the field of tissue culture rapid propagation technology, and more specifically to a tissue culture rapid propagation medium combination and rapid propagation method for Lithospermum macrocephala. Background Technology

[0002] *Mertensia sibirica* (L.) G. Don, belonging to the genus *Mertensia* in the family Boraginaceae, is a perennial herb with slender or short rhizomes. Basal leaves are clustered, while cauline leaves are alternate. The inflorescence is a cyme, usually forming a small panicle at the upper part of the stem. It lacks bracts, and the corolla is funnel-shaped, usually blue or pale blue. The nutlets are tetrahedral. *Mertensia sibirica* has a high annual growth rate and produces abundant blue flowers. Its seeds contain 22.8% gamma-linolenic acid (GLA) in their fatty acid content, giving it unique ornamental and economic value. In the wild, it has a narrow natural distribution only on high-altitude slopes above 2200m, and its populations are sparse.

[0003] There are currently no reports on the propagation techniques of Lithospermum macrocephalum. In order to make reasonable use of this promising plant, plant tissue culture propagation can meet the demand for rapid propagation of seedlings in a short period of time.

[0004] Therefore, whether or not a combination of tissue culture medium and rapid propagation method for Lithospermum macrocephala can be provided is a problem that urgently needs 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 medium combination for Lithospermum macrocephala and a rapid propagation method thereof.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A combination of tissue culture and rapid propagation media for Lithospermum macrocephala, comprising multiple media including primary culture medium, subculture medium, and rooting medium;

[0008] The primary culture medium consisted of: 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 consisted of: MS, 0.5–1.0 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0010] The rooting medium consists of 1 / 8 MS and 0.1 mg / L IBA.

[0011] Preferred media include primary culture medium, subculture medium, and rooting medium;

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

[0013] The subculture medium consisted of MS, 0.8 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar.

[0014] The rooting medium consists of 1 / 8 MS and 0.1 mg / L IBA.

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

[0016] (1) Disinfecting the flower stalk;

[0017] (2) Primary culture: Cut off both ends of the sterilized flower stalk, cut the flower stalk into small segments along the cross section, inoculate the flower stalk into the primary culture medium, and obtain callus tissue and adventitious buds;

[0018] (3) Subculture: The adventitious buds from step (2) were cut and cultured in subculture proliferation medium.

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

[0020] (5) Hardening off seedlings: The test-tube seedlings cultured in step (4) are placed in the hardening off room to harden off seedlings, and seedlings are obtained and ready for transplanting.

[0021] (6) Transplanting.

[0022] Preferred method: Step (1) is as follows: Select tender flower stalks, soak them in laundry detergent 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, then disinfect them with 0.1% HgCl2 for 1 minute, rinse them with sterile water five times, and dry them with sterile filter paper for later use.

[0023] Preferred method: Step (2) specifically involves: using a sterile scalpel to cut off both ends of the disinfected flower stalk, cutting the flower stalk into 1.0cm segments along the cross-section, and inoculating the flower stalk into the primary culture medium for 35-50 days to obtain callus tissue and adventitious buds; the culture conditions are: 22-25℃, light intensity 2000-2500Lux, and photoperiod of 12h / d.

[0024] Preferred: Step (3) specifically involves cutting the adventitious buds from step (2) and then inoculating them into a subculture proliferation medium at 22-25°C, with a light intensity of 2000-2500 Lux and a photoperiod of 12h / d for 20-25 days.

[0025] Preferred step (4) specifically involves: individually inoculating the adventitious buds that have reached a height of 5cm obtained in step (3) into sterile water for culture at 22-25℃, light intensity of 2000-2500Lux, and photoperiod of 12h / d to obtain test-tube seedlings;

[0026] or,

[0027] Individual adventitious buds that reached a height of 5cm in step (3) were inoculated into 1 / 8MS culture medium and cultured at 22-25℃, light intensity of 2000-2500Lux, and photoperiod of 12h / d to obtain test-tube seedlings;

[0028] or,

[0029] Individual adventitious buds that reached a height of 5cm in step (3) were inoculated into rooting medium and cultured at 22-25℃, light intensity of 2000-2500Lux, and photoperiod of 12h / d to obtain test-tube seedlings.

[0030] Preferred step (5) is 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.

[0031] Preferred: Step (6) specifically involves: taking out the seedlings cultivated in step (5) and transplanting them into the test tube seedling transplanting substrate, placing them in a small arched shed in the greenhouse, maintaining the humidity of the arched shed at 80% to 85% and the temperature at 20 to 25°C, gradually opening the small arched shed one week after transplanting, and entering regular management after 30 days;

[0032] The substrate for transplanting test-tube seedlings is peat moss, perlite, and vermiculite in a mass ratio of 3:1:1. Before transplanting, the substrate is disinfected with a 1000-fold dilution of 50% carbendazim wettable powder.

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

[0034] As can be seen from the above technical solution, compared with the prior art, this invention discloses a combination of tissue culture medium and a rapid propagation method for *Lithospermum macrocephalum*. The technical effect achieved is that the tissue culture rapid propagation method for *Lithospermum macrocephalum* provided by this invention utilizes plant tissue culture technology, using flower stalks as explants, which are easy to obtain. The selected subculture and rooting media solve the problem of large-scale cultivation of *Lithospermum macrocephalum*. In particular, during the rooting culture process, the modified 1 / 8MS nutrient solution with added auxin eliminates the step of preparing the culture medium, enabling *Lithospermum macrocephalum* to root with a rooting rate of up to 90%. There is no need to wash the roots with the culture medium during transplanting, simplifying the operation. This method simplifies the tissue culture procedure, saves production costs, and is easy to implement, which is of great significance for the large-scale seedling production of *Lithospermum macrocephalum*. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This invention discloses a tissue culture rapid propagation medium combination for Lithospermum macrocephala and its rapid propagation method.

[0037] Example 1

[0038] A combination of tissue culture and rapid propagation media for Lithospermum macrocephala, including primary culture medium and subculture medium;

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

[0040] The subculture medium consisted of: MS, 0.5 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0041] A rapid breeding method includes the following steps:

[0042] (1) Flower scape disinfection: Select tender flower scapes, soak them in laundry detergent for 10 minutes, 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, then disinfect them with 75% alcohol solution for 30 seconds, rinse them twice with sterile water, then disinfect them with 0.1% HgCl2 for 1 minute, rinse them five times with sterile water, and dry them with sterile filter paper for later use.

[0043] (2) Primary culture: Use a sterile scalpel to cut off both ends of the disinfected flower stalk, cut the flower stalk into 1.0cm segments along the cross section, and inoculate the flower stalk into MS medium with pH 5.8-6.2 and culture for 35-50 days to obtain callus and adventitious shoots;

[0044] The cultivation conditions are: 22–25℃, light intensity 2000–2500 Lux, and photoperiod 12 h / d;

[0045] (3) Subculture: After cutting the adventitious buds in step (2), they were respectively inoculated into subculture proliferation medium. MS medium was used as the basic medium, and 0.5 mg / L of 6-benzylaminopurine (6-BA), 0.01 mg / L of naphthaleneacetic acid (NAA), 30 g / L of sucrose and 6 g / L of agar were added to prepare a subculture medium with a pH of 5.8 to 6.2. The culture conditions were the same as 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: Individual adventitious buds that have reached a height of 5cm obtained in step (3) are inoculated into sterile water for culture under the same conditions as in step (2). After 20 days, the rooting rate can reach 20%, and test-tube seedlings are obtained.

[0047] (5) Hardening off seedlings: When the roots of the test-tube seedlings cultured in step (4) grow to 1-2cm, place them in the hardening off room for 3 days to harden off seedlings and prepare for transplanting.

[0048] (6) Transplanting: The substrate for transplanting test-tube seedlings is peat moss: perlite: vermiculite = 3:1:1. Before transplanting, the substrate 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 substrate for transplanting test-tube seedlings. They are placed in a small arched shed in the greenhouse. The humidity of the arched shed is maintained at 80% to 85% and the temperature at 20 to 25℃. The small arched shed is gradually opened one week after transplanting. After 30 days, it enters regular management.

[0049] Example 2

[0050] A combination of tissue culture and rapid propagation media for Lithospermum macrocephala, comprising one or more of primary culture medium, subculture medium, and rooting medium;

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

[0052] The subculture medium consisted of MS, 0.8 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar.

[0053] The rooting medium consists of 1 / 8 MS and 0.1 mg / L IBA.

[0054] A rapid breeding method includes the following steps:

[0055] (1) Flower scape disinfection: Select tender flower scapes, soak them in laundry detergent for 10 minutes, 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, then disinfect them with 75% alcohol solution for 30 seconds, rinse them twice with sterile water, then disinfect them with 0.1% HgCl2 for 1 minute, rinse them five times with sterile water, and dry them with sterile filter paper for later use.

[0056] (2) Primary culture: Use a sterile scalpel to cut off both ends of the sterilized flower stalk, cut the flower stalk into 1.0cm segments along the cross section, and inoculate the flower stalk into MS medium with pH 5.8-6.2 and culture for 35-50 days to obtain callus and adventitious shoots;

[0057] The cultivation conditions were: 22–25℃, light intensity 2000–2500 Lux, and photoperiod 12 h / d.

[0058] (3) Subculture: After cutting the adventitious buds in step (2), they were respectively inoculated into subculture proliferation medium. MS medium was used as the basic medium, and 0.8 mg / L of 6-benzylaminopurine (6-BA), 0.01 mg / L of naphthaleneacetic acid (NAA), 30 g / L of sucrose and 6 g / L of agar were added to prepare a subculture medium with a pH of 5.8 to 6.2. The culture conditions were the same as 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: Individual adventitious buds that have reached a height of 5cm in step (3) are inoculated into rooting medium of 1 / 8MS + 0.1mg / LIBA and cultured under the same conditions as in step (2). After 20 days, the rooting rate can reach 90%, and test-tube seedlings are obtained.

[0060] (5) Hardening off seedlings: When the roots of the test-tube seedlings cultured in step (4) grow to 1-2cm, place them in the hardening off room for 3 days to harden off seedlings and prepare for transplanting.

[0061] (6) Transplanting: The substrate for transplanting test-tube seedlings is peat moss: perlite: vermiculite = 3:1:1. Before transplanting, the substrate 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 substrate and placed in a small arched shed in the greenhouse. The humidity of the arched shed is maintained at 80% to 85% and the temperature at 20 to 25℃. The small arched shed is gradually opened one week after transplanting, and regular management begins 30 days later.

[0062] Example 3

[0063] A combination of tissue culture and rapid propagation media for Lithospermum macrocephala, including primary culture medium and subculture medium;

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

[0065] The subculture medium consisted of: MS, 1.0 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar;

[0066] A rapid breeding method includes the following steps:

[0067] (1) Flower scape disinfection: Select tender flower scapes, soak them in laundry detergent for 10 minutes, 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, then disinfect them with 75% alcohol solution for 30 seconds, rinse them twice with sterile water, then disinfect them with 0.1% HgCl2 for 1 minute, rinse them five times with sterile water, and dry them with sterile filter paper for later use.

[0068] (2) Primary culture: Use a sterile scalpel to cut off both ends of the sterilized flower stalk, cut the flower stalk into 1.0cm segments along the cross section, and inoculate the flower stalk into MS medium with pH 5.8-6.2 and culture for 35-50 days to obtain callus and adventitious shoots;

[0069] The cultivation conditions are: 22–25℃, light intensity 2000–2500 Lux, and photoperiod 12 h / d;

[0070] (3) Subculture: After cutting the adventitious buds in step (2), they were respectively transferred to subculture proliferation medium. MS medium was used as the basic medium, and 1.0 mg / L of 6-benzylaminopurine (6-BA), 0.01 mg / L of naphthaleneacetic acid (NAA), 30 g / L of sucrose and 6 g / L of agar were added to prepare a subculture medium with a pH of 5.8 to 6.2. The culture conditions were the same as 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: Individual adventitious buds that have reached a height of 5cm in step (3) are inoculated into 1 / 8MS culture medium and cultured under the same conditions as (2). After 20 days, the rooting rate can reach 75%, and test-tube seedlings are obtained.

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

[0073] (6) Transplanting: The substrate for transplanting test-tube seedlings is peat moss: perlite: vermiculite = 3:1:1. Before transplanting, the substrate 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 substrate and placed in a small arched shed in the greenhouse. The humidity of the arched shed is maintained at 80% to 85% and the temperature at 20 to 25℃. The small arched shed is gradually opened one week after transplanting, and regular management begins 30 days later.

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

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

[0076] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A tissue culture medium composition for rapid propagation of *Lithospermum macrocephala*, characterized in that, The flower stalk was used as the explant; the culture medium consisted of primary culture medium, subculture medium and rooting medium. The primary culture medium consisted of: MS, 1.5 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar. The subculture medium consisted of 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 was 1 / 8 MS, 0.1 mg / L IBA.

2. The culture medium combination as described in claim 1, characterized in that, The primary culture medium consisted of: MS, 1.5 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar. The subculture medium consisted of MS, 0.8 mg / L 6-BA, 0.01 mg / L NAA, 30 g / L sucrose, and 6 g / L agar. The rooting medium was 1 / 8 MS, 0.1 mg / L IBA.

3. A method for rapid propagation of *Lithospermum macrocephalum* using the culture medium combination described in claim 1 or 2, characterized in that, Includes the following steps: (1) Disinfecting flower stalks; (2) Primary culture: Cut off both ends of the sterilized flower stalk, cut the flower stalk into small segments along the cross section, inoculate the flower stalk into the primary culture medium, and obtain callus tissue and adventitious buds; (3) Subculture: After cutting the adventitious buds in step (2), they were cultured in subculture medium. (4) Rooting culture: Individual adventitious buds obtained in step (3) are inoculated into rooting culture medium to obtain test-tube seedlings; (5) Hardening off seedlings: The test-tube seedlings cultured in step (4) are placed in the hardening off room to harden off seedlings and prepare for transplanting; (6) Transplanting.

4. The rapid propagation method as described in claim 3, characterized in that, Step (1) is as follows: Select tender flower stalks, soak them in laundry detergent for 10 minutes, rinse them with tap water, dry them with filter paper, rinse them once with sterile water, disinfect them with 75% alcohol solution for 30 seconds, rinse them twice with sterile water, then disinfect them with 0.1% HgCl2 for 1 minute, rinse them five times with sterile water, and dry them with sterile filter paper for later use.

5. The rapid propagation method as described in claim 4, characterized in that, Step (2) is as follows: Use a sterile scalpel to cut off both ends of the disinfected flower stalk, cut the flower stalk into 1.0 cm segments along the cross section, and inoculate the flower stalk into the primary culture medium and culture for 35-50 days to obtain callus and adventitious buds; the culture conditions are: 22-25℃, light intensity 2000-2500 Lux, and photoperiod of 12 h / d.

6. The rapid propagation method as described in claim 5, characterized in that, Step (3) is as follows: after cutting the adventitious buds in step (2), they are respectively inoculated into the subculture proliferation medium, and cultured for 20 to 25 days at 22~25℃, light intensity of 2000~2500 Lux, and photoperiod of 12h / d.

7. The rapid propagation method as described in claim 6, characterized in that, Step (4) specifically involves individually inoculating the adventitious buds that have reached a height of 5 cm in step (3) into a rooting medium and culturing them at 22-25℃, with a light intensity of 2000-2500 Lux and a photoperiod of 12 h / d to obtain test-tube seedlings.

8. The rapid propagation method as described in claim 7, characterized in that, Step (5) is 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 as described in claim 8, characterized in that, Step (6) specifically involves taking out the seedlings cultivated in step (5) and transplanting them into the test tube seedling transplanting substrate. The seedlings are placed in a small arched shed in the greenhouse. The humidity of the arched shed is maintained at 80%~85% and the temperature at 20~25℃. The small arched shed is gradually opened one week after transplanting, and the seedlings are put into regular management after 30 days. The substrate for transplanting the test-tube seedlings consisted of peat moss, perlite, and vermiculite in a mass ratio of 3:1:

1. Before transplanting, the substrate was disinfected with a 1000-fold dilution of 50% carbendazim wettable powder.

10. The application of the culture medium combination of *Lithospermum macrocephalum* as described in claim 1 or 2, or the rapid propagation method of *Lithospermum macrocephalum* as described in any one of claims 3 to 9, in the tissue culture of *Lithospermum macrocephalum*.