Induction method of gingko embryonic stem cells

Abstract

The invention relates to an induction method of gingko embryonic stem cells, which comprises the following steps: (1) collecting tender branches of gingko, cleaning, soaking and disinfecting; (2) removing leaves and xylem, cutting the remaining part into small blocks, inoculating the small blocks on a culture medium for culturing, and enabling a cambium to be in contact with the culture medium; calluses start to grow on the parts, in contact with the culture medium, around the small branches; (3) carrying out subculture on the calluses for 2-3 generations to form granular calluses with loose structures, and the culture medium for subculture is different from the culture medium in the step (2); (4) inoculating the embryogenic stem cells of the calluses into an MS liquid culture medium, and carrying out suspension amplification culture; and (5) collecting the ginkgo biloba embryogenic stem cells subjected to amplification culture.

Description

Technical Field

[0001] The invention belongs to the technical field of plant stem cell culture, and particularly relates to an induction culture method for ginkgo embryonic stem cells. Technical Background

[0002] Ginkgoaceae is a deciduous tree in the Ginkgoaceae family, Ginkgoales. It has only one genus and one species, Ginkgo, native to China. The leaves of Ginkgoaceae plants are fan-shaped, scattered on long branches and clustered on short branches. The flowers are unisexual and dioecious, and the seeds are drupe-shaped.

[0003] Ginkgo contains a variety of medicinal secondary metabolites, including flavonoids and their glycosides, terpene lactones, organic acids, alkaloids, amino acids, isopentenols, steroids, etc. Among them, terpene lactones of ginkgo are natural and specific antagonists of platelet-activating factor receptors, and are also excellent natural medicines for the treatment and prevention of cardiovascular and cerebrovascular diseases. Because of the large market demand, traditional planting has many shortcomings, such as long plant growth cycle, low yield, susceptibility to climate and environmental conditions, and difficulty in regulation, which restricts the production capacity of the antagonist drug. At the same time, the traditional extraction, separation and purification methods cannot meet the requirements, and the chemical total synthesis steps are complicated and costly, and do not have industrial production value. Therefore, scientists have turned their attention to modern biotechnology. Biotechnology such as plant cell culture can well regulate the growth environment and metabolism of cells, is not affected by the environment and climate, and combines large-scale bioreactor technology to make industrial fermentation possible. Plant tissue culture technology is the application of cell culture technology in plants. Compared with chemical synthesis, plant tissue culture has the characteristics of environmental friendliness, low investment and high economic benefits, and its application prospects are broad.

[0004] The process of plant tissue culture technology mainly includes collecting explants and sterilizing them. The explants are damaged by physical stimulation such as knife cutting. The somatic cells at the damaged explants will dedifferentiate and form callus tissue. After continuous subculture of the callus tissue, a stably cultured callus cell line is obtained. Select callus tissue with loose texture, and subculture it in liquid culture medium for suspension culture. Using fermentation technology, a large amount of cell culture can be obtained, and then the required natural secondary metabolites can be obtained. Although scholars began to study ginkgo suspension cells as early as the last century, the results were minimal. The main defect of ginkgo cell culture is that the content of ginkgo terpene lactones after cell culture is low, which cannot meet the requirements of large-scale industrial production.

[0005] Studies have found that in the stem cells of the vascular cambium of the ginkgo trunk, although the growth of the vascular tissue in the trunk slows down, the increase in the cross-sectional area of ​​the trunk is still at a high level, indicating that the stem cells of the cambium of the ginkgo trunk still have a strong and continuous division ability. Based on this characteristic of ginkgo, a ginkgo stem cell culture system can be established to provide a new culture method for inducing ginkgo embryonic stem cells, increase the diversity of ginkgo stem cell induction methods, and at the same time increase the content of ginkgo terpene lactones.

[0006] CN102459572A discloses a method for culturing and isolating stem cells derived from the cambium of a Ginkgoaceae plant. The tissue with cambium is obtained from a Ginkgoaceae plant; the cambium tissue is cultured in a culture medium; cells are separated from the cambium to obtain stem cells derived from the cambium. The stem cells are separated into an undifferentiated state without a dedifferentiation process, so that the cell growth rate and growth pattern can be maintained stably without variation during long-term culture and can be cultured in large quantities, thereby being useful.

[0007] CN107119007A discloses a method for inducing and identifying stem cells of the root cambium of ginkgo biloba. The ginkgo stem cells grow and reproduce rapidly and have a stable shape during long-term culture, and can be cultured in large quantities. The stem cells induced and cultured by the method are round in shape and contain small and numerous vacuoles. They mostly exist in a single cell state during suspension culture, and have more types of intracellular terpene lactones and higher content.

[0008] CN116445393A discloses a plant stem cell line and its establishment method, including a) stem cell culture; b) stem cell screening; c) stem cell subculture; after each generation of culture, before the next subculture, stem cell screening is performed to obtain a plant stem cell cluster from a clone; d) stem cell screening; e) stem cell line establishment, after each generation of culture, before the next subculture, stem cell screening is performed; f) stem cell line domestication to obtain one or more plant stem cell lines with stable traits, stable division and rapid division. Summary of the invention

[0009] One of the purposes of the present invention is to establish a new ginkgo stem cell culture system according to the characteristics of ginkgo, so as to increase the diversity of ginkgo stem cell induction methods.

[0010] A method for inducing Ginkgo embryonic stem cells comprises the following steps:

[0011] (1) Collect young and tender branches of Ginkgo biloba, clean, soak and disinfect them;

[0012] (2) removing leaves and wood, cutting the remaining parts into small pieces, inoculating them on a culture medium, and culturing them with the cambium in contact with the culture medium; callus tissue begins to grow around the small branches in contact with the culture medium;

[0013] (3) subculturing the callus tissue to form a loosely structured granular callus tissue after 2-3 generations of subculturing, and the subculturing medium is different from that in the above step (2);

[0014] (4) The callus embryonic stem cells are inoculated into MS liquid culture medium for suspension expansion culture, and the expanded Ginkgo embryonic stem cells are collected.

[0015] The cleaning in step (1) is performed with deionized water for 3-5 times; the soaking is performed with anhydrous ethanol for 1-12 hours; the disinfection is performed with a hydrogen peroxide solution or a sodium hypochlorite solution for 3-5 times, wherein the concentration of hydrogen peroxide in the hydrogen peroxide solution is 10-20wt%, and the concentration of the sodium hypochlorite solution is 1-5%.

[0016] In step (2), cut the remaining part after removing the leaves and wood into 1-2 cm 2 The callus culture time is 10-20 days, preferably 12-15 days.

[0017] In steps (2) and (3), the culture medium comprises one or more of MS (Murashige and Skoog medium), 2,4-D (2,4-dichlorophenoxyacetic acid), IBA (indolebutyric acid), GA3 (gibberellic acid), 6-BA (6-benzyladenine), NAA (naphthaleneacetic acid), and KT (6-furfurylaminopurine); preferably comprises MS+2,4-D+IBA+GA3, or MS+6-BA+NAA+KT;

[0018] More preferably, it comprises: MS+2,4-D (1.0-1.5 mg / L)+IBA (1.0-2.0 mg / L)+GA3 (0.1-0.5 mg / L), or MS+6-BA (1.0-1.5 mg / L)+NAA (0.5-1.0 mg / L)+KT (0.5-1.0 mg / L); the culture medium also contains agar and sucrose, wherein the amount of agar is 5-20 g / L, and the amount of sucrose is 10-50 g / L; the pH value of the culture medium is 5-7, more preferably 5-6.

[0019] The culture medium in step (4) is MS+0.5 mg / L 2,4-D+2.0 mg / L 6-BA+0.5 mg / L KT.

[0020] The callus induction rate in step (3) can reach more than 60%, and the callus induction rate in step (4) can reach more than 80%.

[0021] In steps (2) to (4), the culture temperature is 25±2°C, the illumination time is 8-16h / d, and the illumination intensity is 30-80μmol·m -2 ·s-1 .

[0022] The collection method in step (4) is one of filtration, suction filtration, sedimentation and centrifugal separation.

[0023] The content of ginkgo terpene lactones in the ginkgo embryonic stem cells prepared by the method is greater than 0.63 wt%, more preferably greater than 0.65 wt%.

[0024] Beneficial technical effects of the present invention:

[0025] The present invention adopts the stable culture of cambium stem cells collected from ginkgo branches, which significantly improves the yield of stem cells. The content of terpene lactones in the formed stem cells is also higher than that of terpene lactones extracted from natural cells, which is conducive to the industrial production of related drugs. DETAILED DESCRIPTION

[0026] In order to better understand the technical solution of the present invention, the content of the present invention includes but is not limited to the specific implementation methods described below, and similar technologies and methods should be considered to be within the scope of protection of the present invention.

[0027] The present invention is further described in detail below with specific implementation modes.

[0028] Example 1

[0029] (1) Collection and processing of ginkgo branches: Collect young ginkgo branches, rinse with clean water to remove dust and other stains on the surface, soak with detergent for 15 minutes, and then wash with clean water 5 times;

[0030] (2) Explant disinfection and sterilization: On a clean bench, soak the ginkgo branches in 75% ethanol for 30 seconds, rinse with sterile water three times, soak in sodium hypochlorite aqueous solution for 15 minutes, rinse with sterile water six times, and dry the surface moisture of the branches with sterile paper for later use;

[0031] (3) Primary culture of explants: Cut off the wounds with mechanical damage, remove the xylem of the branches with a sterile knife, and then cut the branches with the xylem removed into small pieces of 1 square centimeter, inoculate them on MS+1.0mg / L 2,4-D+2.0mg / LIBA+0.3mg / LGA3 medium for culture, with the cambium in contact with the medium; after 15 days, small yellow particles begin to grow around the small branches in contact with the medium, which are callus tissue, and the callus induction rate can reach 65%;

[0032] (4) Subculture of callus tissue: The induced callus tissue was removed from the explant and transferred to MS+1.5mg / L6-BA+0.5mg / LNAA+0.8mg / LKT medium for subculture. After 2-3 generations of subculture, embryonic callus tissue was obtained. The expanded cultured Ginkgo embryonic stem cells were collected. The induction rate of embryonic callus tissue reached 80%;

[0033] The above culture medium contained 30 g / L sucrose and 8 g / L agar, pH = 5.8, culture temperature was 25 ± 2 °C, light duration was 14 h / d, light intensity was 55 μmol·m -2 ·s -1 .

[0034] Example 2

[0035] (1) Collection and processing of ginkgo branches: Collect young ginkgo branches, rinse with clean water to remove dust and other stains on the surface, soak with detergent for 15 minutes, and then wash with clean water 5 times;

[0036] (2) Explant disinfection and sterilization: On a clean bench, soak the ginkgo branches in 75% ethanol for 30 seconds, rinse with sterile water three times, soak in sodium hypochlorite aqueous solution for 15 minutes, rinse with sterile water six times, and dry the surface moisture of the branches with sterile paper for later use;

[0037] (3) Primary culture of explants: Cut off the wounds with mechanical damage, remove the xylem of the branches with a sterile knife, and then cut the branches with the xylem removed into small pieces of 1 square centimeter, inoculate them on MS+1.5mg / L 2,4-D+1.0mg / LIBA+0.2mg / LGA3 medium, and culture them with the cambium in contact with the medium. After 15 days, small yellow particles begin to grow around the small branches in contact with the medium, which is callus tissue. The callus induction rate can reach 70%;

[0038] (4) Subculture of callus: The induced callus was removed from the explant and transferred to MS+1.0mg / L6-BA+0.5mg / LNAA+1.0mg / LKT medium for subculture. After 2-3 generations of subculture, embryonic callus was obtained. The expanded cultured Ginkgo embryonic stem cells were collected. The induction rate of embryonic callus reached 85%;

[0039] The above culture medium contained 30 g / L sucrose and 8 g / L agar, pH = 5.8, culture temperature was 25 ± 2 °C, light duration was 14 h / d, light intensity was 55 μmol·m -2 ·s -1 .

[0040] Example 3

[0041] (1) Collection and processing of ginkgo branches: Collect young ginkgo branches, rinse with clean water to remove dust and other stains on the surface, soak with detergent for 15 minutes, and then wash with clean water 5 times;

[0042] (2) Explant disinfection and sterilization: On a clean bench, soak the ginkgo branches in 75% ethanol for 30 seconds, rinse with sterile water three times, soak in sodium hypochlorite aqueous solution for 15 minutes, rinse with sterile water six times, and dry the surface moisture of the branches with sterile paper for later use;

[0043] (3) Primary culture of explants: Cut off the wounds with mechanical damage, remove the xylem of the branches with a sterile knife, and then cut the branches with the xylem removed into small pieces of 1 square centimeter, inoculate them on MS+1.2mg / L 2,4-D+1.5mg / LIBA+0.5mg / LGA3 medium for culture, with the cambium in contact with the medium. After 15 days, small yellow particles begin to grow around the small branches in contact with the medium, which are callus tissue. The callus induction rate can reach 60%;

[0044] (4) Subculture of callus tissue: The induced callus tissue was removed from the explant and transferred to MS+1.0mg / L6-BA+1.0mg / LNAA+0.5mg / LKT medium for subculture. After 2-3 generations of subculture, embryonic callus tissue was obtained. The expanded cultured Ginkgo embryonic stem cells were collected. The induction rate of embryonic callus tissue reached 80%;

[0045] The above culture medium contained 30 g / L sucrose and 8 g / L agar, pH = 5.8, culture temperature was 25 ± 2 °C, light duration was 14 h / d, light intensity was 55 μmol·m -2 ·s -1 .

[0046] Example 4

[0047] (1) Collection and processing of ginkgo branches: Collect young ginkgo branches, rinse with clean water to remove dust and other stains on the surface, soak with detergent for 15 minutes, and then wash with clean water 5 times;

[0048] (2) Explant disinfection and sterilization: On a clean bench, soak the ginkgo branches in 75% ethanol for 30 seconds, rinse with sterile water three times, soak in sodium hypochlorite aqueous solution for 15 minutes, rinse with sterile water six times, and dry the surface moisture of the branches with sterile paper for later use;

[0049] (3) Primary culture of explants: Cut off the wounds with mechanical damage, remove the xylem of the branches with a sterile knife, and then cut the branches with the xylem removed into small pieces of 1 square centimeter, inoculate them on MS+1.0mg / L 2,4-D+1.5mg / LIBA+0.5mg / LGA3 medium for culture, with the cambium contacting the medium. After 15 days, small yellow particles begin to grow around the small branches in contact with the medium, which is callus tissue, and the callus induction rate reaches 65%;

[0050] (4) Subculture of callus: The induced callus was removed from the explant and transferred to MS+1.2mg / L6-BA+0.8mg / L NAA+1.0mg / L KT medium for subculture. After 2-3 generations of subculture, embryonic callus was obtained. The expanded cultured Ginkgo embryonic stem cells were collected. The induction rate of embryonic callus reached 85%;

[0051] The above culture medium contained 30 g / L sucrose and 8 g / L agar, pH = 5.8, culture temperature was 25 ± 2 °C, light duration was 14 h / d, light intensity was 55 μmol·m -2 ·s -1 .

[0052] Example 5

[0053] (1) Collection and processing of ginkgo branches: Collect young ginkgo branches, rinse with clean water to remove dust and other stains on the surface, soak with detergent for 15 minutes, and then wash with clean water 5 times;

[0054] (2) Explant disinfection and sterilization: On a clean bench, soak the ginkgo branches in 75% ethanol for 30 seconds, rinse with sterile water three times, soak in sodium hypochlorite aqueous solution for 15 minutes, rinse with sterile water six times, and dry the surface moisture of the branches with sterile paper for later use;

[0055] (3) Primary culture of explants: Cut off the wounds with mechanical damage, remove the xylem of the branches with a sterile knife, and then cut the branches with the xylem removed into small pieces of 1 square centimeter, inoculate them on MS+1.2mg / L 2,4-D+2.0mg / LIBA+0.1mg / LGA3 medium, and culture them with the cambium in contact with the medium. After 15 days, small yellow particles begin to grow around the small branches in contact with the medium, which is callus tissue, and the callus induction rate reaches 70%;

[0056] (4) Subculture of callus tissue: The induced callus tissue was removed from the explant and transferred to MS+1.5mg / L6-BA+1.0mg / L NAA+0.5mg / L KT medium for subculture. After 2-3 generations of subculture, embryonic callus tissue was obtained. The expanded cultured Ginkgo embryonic stem cells were collected, and the induction rate of embryonic callus tissue reached 85%;

[0057] The above culture medium contained 30 g / L sucrose and 8 g / L agar, pH = 5.8, culture temperature was 25 ± 2 °C, light duration was 14 h / d, light intensity was 55 μmol·m -2 ·s -1 .

[0058] Comparative Example 1

[0059] (1) Collection and processing of ginkgo branches: Collect young ginkgo branches, rinse with clean water to remove dust and other stains on the surface, soak with detergent for 15 minutes, and then wash with clean water 5 times;

[0060] (2) Explant disinfection and sterilization: On a clean bench, soak the ginkgo branches in 75% ethanol for 30 seconds, rinse with sterile water three times, soak in sodium hypochlorite aqueous solution for 15 minutes, rinse with sterile water six times, and dry the surface moisture of the branches with sterile paper for later use;

[0061] (3) Primary culture of explants: Cut off the wounds with mechanical damage, remove the xylem of the branches with a sterile knife, and then cut the branches with the xylem removed into small pieces of 1 square centimeter. Inoculate them on MS medium and culture them with the cambium in contact with the medium. After 15 days, small yellow particles begin to grow around the small branches in contact with the medium. These are callus tissues. The callus induction rate reaches 70%;

[0062] (4) Subculture of callus tissue: The induced callus tissue is removed from the explant and transferred to MS medium for subculture. After 2-3 generations of subculture, embryonic callus tissue can be obtained. The expanded cultured Ginkgo embryonic stem cells are collected, and the induction rate of embryonic callus tissue reaches 85%;

[0063] The above culture medium contained 30 g / L sucrose and 8 g / L agar, pH = 5.8, culture temperature was 25 ± 2 °C, light duration was 14 h / d, light intensity was 55 μmol·m -2 ·s -1 .

[0064] Comparative Example 2

[0065] (1) Collection and processing of ginkgo roots: Ginkgo roots were collected, rinsed with clean water to remove dust and other stains on the surface, soaked in detergent for 15 minutes, and then washed with clean water 5 times;

[0066] (2) Explant disinfection and sterilization: On a clean bench, soak the roots of ginkgo in 75% ethanol for 30 seconds, rinse with sterile water three times, soak in sodium hypochlorite aqueous solution for 15 minutes, rinse with sterile water six times, and dry the surface moisture of the roots with sterile paper for later use;

[0067] (3) Primary culture of explants: Cut off the wounds with mechanical damage, remove the xylem of the roots with a sterile knife, and then cut the roots with the xylem removed into small pieces of 1 cm2, and inoculate them on MS+1.0 mg / L 2,4-D+2.0 mg / LIBA+0.3 mg / LGA3 culture medium, with the cambium in contact with the culture medium; after 15 days, small yellow particles begin to grow around the small root pieces in contact with the culture medium, which are callus tissue, and the callus induction rate can reach 45%;

[0068] (4) Subculture of callus: The induced callus was removed from the explant and transferred to MS+1.5mg / L6-BA+0.5mg / LNAA+0.8mg / LKT medium for subculture. After 2-3 generations of subculture, embryonic callus was obtained. The expanded cultured Ginkgo embryonic stem cells were collected. The induction rate of embryonic callus reached 70%.

[0069] The above culture medium contained 30 g / L sucrose and 8 g / L agar, pH = 5.8, culture temperature was 25 ± 2 °C, light duration was 14 h / d, light intensity was 55 μmol·m -2 ·s -1 .

[0070] Comparative Example 3

[0071] (1) Collection and processing of ginkgo roots: Ginkgo roots were collected, rinsed with clean water to remove dust and other stains on the surface, soaked in detergent for 15 minutes, and then washed with clean water 5 times;

[0072] (2) Explant disinfection and sterilization: On a clean bench, soak the roots of ginkgo in 75% ethanol for 30 seconds, rinse with sterile water three times, soak in sodium hypochlorite aqueous solution for 15 minutes, rinse with sterile water six times, and dry the surface moisture of the roots with sterile paper for later use;

[0073] (3) Primary culture of explants: Cut off the wounds with mechanical damage, remove the xylem of the roots with a sterile knife, and then cut the roots with the xylem removed into small pieces of 1 square centimeter, inoculate them on MS+1.5mg / L 2,4-D+1.0mg / LIBA+0.2mg / LGA3 medium, and culture them with the cambium in contact with the medium. After 15 days, small yellow particles begin to grow around the small root pieces in contact with the medium, which are callus tissue. The callus induction rate can reach 40%;

[0074] (4) Subculture of callus tissue: The induced callus tissue was removed from the explant and transferred to MS + 1.0 mg / L 6-

[0075] Subculture was performed on BA+0.5mg / LNAA+1.0mg / LKT medium. After 2-3 generations of subculture, embryonic callus tissue was obtained. The embryonic stem cells of Ginkgo biloba in expanded culture were collected. The induction rate of embryonic callus tissue reached 70%.

[0076] The above culture medium contained 30 g / L sucrose and 8 g / L agar, pH = 5.8, culture temperature was 25 ± 2 °C, light duration was 14 h / d, light intensity was 55 μmol·m -2 ·s -1 .

[0077] Comparative Example 4

[0078] (1) Collection and processing of ginkgo roots: Ginkgo roots were collected, rinsed with clean water to remove dust and other stains on the surface, soaked in detergent for 15 minutes, and then washed with clean water 5 times;

[0079] (2) Explant disinfection and sterilization: On a clean bench, soak the roots of ginkgo in 75% ethanol for 30 seconds, rinse with sterile water three times, soak in sodium hypochlorite aqueous solution for 15 minutes, rinse with sterile water six times, and dry the surface moisture of the roots with sterile paper for later use;

[0080] (3) Primary culture of explants: Cut off the wounds with mechanical damage, remove the xylem of the roots with a sterile knife, and then cut the roots with the xylem removed into small pieces of 1 cm2. Inoculate them on MS+1.2mg / L 2,4-D+1.5mg / LIBA+0.5mg / LGA3 medium for culture, with the cambium in contact with the medium. After 15 days, small yellow particles begin to grow around the small root pieces in contact with the medium. These are callus tissue, and the callus induction rate can reach 50%;

[0081] (4) Subculture of callus: The induced callus was removed from the explant and transferred to MS+1.0mg / L6-BA+1.0mg / LNAA+0.5mg / LKT medium for subculture. After 2-3 generations of subculture, embryonic callus was obtained. The expanded cultured Ginkgo embryonic stem cells were collected. The induction rate of embryonic callus reached 75%;

[0082] The above culture medium contained 30 g / L sucrose and 8 g / L agar, pH = 5.8, culture temperature was 25 ± 2 °C, light duration was 14 h / d, light intensity was 55 μmol·m -2 ·s -1 .

[0083] Comparative Example 5

[0084] (1) Collection and processing of ginkgo roots: Ginkgo roots were collected, rinsed with clean water to remove dust and other stains on the surface, soaked in detergent for 15 minutes, and then washed with clean water 5 times;

[0085] (2) Explant disinfection and sterilization: On a clean bench, soak the roots of ginkgo in 75% ethanol for 30 seconds, rinse with sterile water three times, soak in sodium hypochlorite aqueous solution for 15 minutes, rinse with sterile water six times, and dry the surface moisture of the roots with sterile paper for later use;

[0086] (3) Primary culture of explants: Cut off the wounds with mechanical damage, remove the xylem of the roots with a sterile knife, and then cut the roots with the xylem removed into small pieces of 1 cm2, and inoculate them on MS+1.0mg / L 2,4-D+1.5mg / LIBA+0.5mg / LGA3 medium for culture, with the cambium in contact with the medium. After 15 days, small yellow particles begin to grow around the small root pieces in contact with the medium, which are callus tissue, and the callus induction rate reaches 45%;

[0087] (4) Subculture of callus tissue: The induced callus tissue was removed from the explant and transferred to MS+1.2mg / L6-BA+0.8mg / L NAA+1.0mg / L KT medium for subculture. After 2-3 generations of subculture, embryonic callus tissue was obtained. The expanded cultured Ginkgo embryonic stem cells were collected. The induction rate of embryonic callus tissue reached 80%;

[0088] The above culture medium contained 30 g / L sucrose and 8 g / L agar, pH = 5.8, culture temperature was 25 ± 2 °C, light duration was 14 h / d, light intensity was 55 μmol·m -2 ·s -1 .

[0089] Comparative Example 6

[0090] (1) Collection and processing of ginkgo roots: Ginkgo roots were collected, rinsed with clean water to remove dust and other stains on the surface, soaked in detergent for 15 minutes, and then washed with clean water 5 times;

[0091] (2) Explant disinfection and sterilization: On a clean bench, soak the roots of ginkgo in 75% ethanol for 30 seconds, rinse with sterile water three times, soak in sodium hypochlorite aqueous solution for 15 minutes, rinse with sterile water six times, and dry the surface moisture of the roots with sterile paper for later use;

[0092] (3) Primary culture of explants: Cut off the wounds with mechanical damage, remove the xylem of the roots with a sterile knife, and then cut the roots with the xylem removed into small pieces of 1 cm2, and inoculate them on MS+1.2mg / L 2,4-D+2.0mg / LIBA+0.1mg / LGA3 medium for culture, with the cambium in contact with the medium. After 15 days, small yellow particles begin to grow around the small root pieces in contact with the medium, which are callus tissue, and the callus induction rate reaches 50%;

[0093] (4) Subculture of callus: The induced callus was removed from the explant and transferred to MS+1.5mg / L6-BA+1.0mg / L NAA+0.5mg / L KT medium for subculture. After 2-3 generations of subculture, embryonic callus was obtained. The expanded cultured Ginkgo embryonic stem cells were collected. The induction rate of embryonic callus reached 75%;

[0094] The above culture medium contained 30 g / L sucrose and 8 g / L agar, pH = 5.8, culture temperature was 25 ± 2 °C, light duration was 14 h / d, light intensity was 55 μmol·m -2 ·s -1 .

[0095] Expansion and collection of Ginkgo biloba embryonic stem cells

[0096] The embryonic callus cells induced in the above embodiment were inoculated into MS + 0.5 mg / L 2,4-D + 2.0 mg / L 6-BA + 0.5 mg / L KT liquid culture medium for suspension expansion culture, wherein amphotericin B and double resistance were also added to the liquid culture medium, and the conical flask containing the culture medium was placed in a constant temperature oscillator, and cultured in the dark at a speed of 110 r / min and a temperature of 22 ° C. Subculture was performed once every 2-3 days, and the embryonic stem cells were collected after being expanded to a certain extent or after 2-3 subcultures. The collected embryonic stem cells should be placed under corresponding temperature conditions as required.

[0097] Neutral red staining was used to identify Ginkgo biloba embryonic stem cells.

[0098] (1) diluting the Ginkgo biloba embryonic stem cells with phosphate buffer, and evenly smearing the diluted embryonic stem cell solution on a glass slide;

[0099] (2) adding 0.1% neutral red dye solution to the embryonic stem cells on the slide to completely cover the embryonic stem cells, and staining for 30 minutes at room temperature;

[0100] (3) After staining, use absorbent paper to absorb the dye outside the stained cells, rinse them with phosphate buffer for 2-3 times, cover with a coverslip, and immediately observe under a microscope.

[0101] (4) The results showed that under the microscope, most Ginkgo embryonic stem cells were round single cells, but there were also a small number of cell clusters with small but numerous vacuoles.

[0102] Detection of the content of ginkgolide in Ginkgo embryonic stem cells by liquid chromatography

[0103] (1) Preparation of reference solution: Accurately weigh appropriate amounts of Ginkgolide A, B, C and bilobalide standard reference solution, place them in a volumetric flask, and dilute to the required concentration of the standard curve with methanol.

[0104] (2) Preparation of test solution: Accurately weigh 1.0 g of sample and place it in a 50 mL beaker. Add 50 mL of 95% ethanol and perform ultrasonic extraction for 1 h. Let stand, cool, filter, and obtain the filtrate.

[0105] Table 1

[0106]

[0107] The test results in Table 1 show that compared with the comparative example, the content of ginkgolide in the ginkgo embryonic stem cells in the example is significantly increased, from which it can be concluded that the newly established culture system shows a significant increase in the content of ginkgolide in the ginkgo embryonic stem cells.

Claims

1. A method for inducing Ginkgo embryonic stem cells, comprising the following steps: (1) Collect young and tender branches of Ginkgo biloba, clean, soak and disinfect them; (2) removing leaves and wood, cutting the remaining parts into small pieces, inoculating them on a culture medium, and culturing them with the cambium in contact with the culture medium; callus tissue begins to grow around the roots of the small pieces that are in contact with the culture medium; (3) subculturing the callus tissue to form a loosely structured granular callus tissue after 2-3 generations of subculturing, wherein the subculturing medium is the same as or different from that in the above step (2); (4) The callus embryonic stem cells are inoculated into MS liquid culture medium for suspension expansion culture, and the expanded Ginkgo embryonic stem cells are collected.

2. The method according to claim 1, characterized in that The cleaning in step (1) is performed with deionized water for 3-5 times; the soaking is performed with anhydrous ethanol for 1-12 hours; the disinfection is performed with a hydrogen peroxide solution or a sodium hypochlorite solution for 3-5 times, wherein the concentration of hydrogen peroxide in the hydrogen peroxide solution is 10-20wt%, and the concentration of the sodium hypochlorite solution is 1-5%.

3. The method according to claim 1, characterized in that In step (2), cut the remaining part after removing the leaves and wood into 1-2 cm 2 The callus culture time is 10-20 days, preferably 12-15 days.

4. The method according to claim 1, characterized in that In steps (2) and (3), the culture medium contains one or more of MS, 2,4-D, IBA, GA3, 6-BA, NAA, and KT; preferably contains MS+2,4-D+IBA+GA3, or MS+6-BA+NAA+KT.

5. The method according to claim 4, characterized in that In steps (2) and (3), the culture medium comprises MS+2,4-D (1.0-1.5 mg / L)+IBA (1.0-2.0 mg / L)+GA3 (0.1-0.5 mg / L), or MS+6-BA (1.0-1.5 mg / L)+NAA (0.5-1.0 mg / L)+KT (0.5-1.0 mg / L); the culture medium also contains agar and sucrose, wherein the amount of agar is 5-20 g / L, and the amount of sucrose is 10-50 g / L; the pH value of the culture medium is 5-7, more preferably 5-6.

6. The method according to claim 1, characterized in that The culture medium in steps (2) and (3) comprises the culture medium in step (4) which is MS+0.5 mg / L 2,4-D+2.0 mg / L 6-BA+0.5 mg / L KT.

7. The method according to claim 1, characterized in that The callus induction rate in step (3) can reach more than 60%, and the callus induction rate in step (4) can reach more than 80%.

8. The method according to claim 1, characterized in that In steps (2) to (4), the culture temperature is 25±2°C, the illumination time is 8-16h / d, and the illumination intensity is 30-80μmol·m -2 ·s -1 .

9. The method according to claim 1, characterized in that The collection method in step (4) is one of filtration, suction filtration, sedimentation and centrifugal separation.

10. The method according to claim 1, characterized in that The content of ginkgolide in the prepared ginkgo embryonic stem cells is greater than 0.63wt%, more preferably greater than 0.65wt%.

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