A method for inducing adventitious roots of panax notoginseng tetraploid

Abstract

This invention relates to a method for inducing tetraploid Panax notoginseng to produce adventitious roots, belonging to the field of biotechnology. The invention includes the following steps: inducing embryogenic callus tissue from immature zygotic embryos of Panax notoginseng; dispersing the embryogenic callus tissue to obtain small-pore embryogenic cell clusters, and culturing them in the dark in Erlenmeyer flasks containing 1-4 mg / mL colchicine; removing the colchicine, and culturing the cell clusters on a solid culture medium to induce tetraploid Panax notoginseng callus tissue; differentiating the tetraploid Panax notoginseng callus tissue on a plate culture medium to obtain cotyledonary embryos; and transferring the cotyledonary embryos to an intermittent submerged bioreactor for culture. This invention utilizes chromosome doubling technology and bioreactor culture technology to obtain a large number of Panax notoginseng adventitious roots, improving the proliferation rate and biomass of Panax notoginseng. Furthermore, the combination of chromosome doubling technology and bioreactor can accelerate the growth cycle of Panax notoginseng adventitious roots, significantly shortening the time from cultivation to harvest and improving production efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a method for inducing adventitious roots of Panax notoginseng tetraploid. BACKGROUND

[0002] Panax notoginseng, also known as Gingseng, belongs to the Araliaceae Panax genus together with Panax ginseng, Panax quinquefolium and Panax japonicus, and is a perennial herbaceous plant. In China, it is mainly distributed in Yunnan and Guangxi, and grows in forests or on slopes at an altitude of 400-1800 meters. Panax notoginseng usually takes more than three years to be harvested, and the harvesting period is from October to December. The main medicinal parts are roots and rhizomes. It is recorded in Shengwu Gangmu Shiyi that "Panax notoginseng is the first in tonifying qi, and Panax notoginseng is the first in tonifying blood". Modern pharmacological studies have shown that Panax notoginseng saponins have the effects of anti-inflammatory, blood lipid regulation, anti-platelet aggregation, anti-tumor, anti-thrombosis, prevention of atherosclerosis and fatty liver, prevention of myocardial infarction, promotion of liver regeneration and angiogenesis, etc. With the gradual exploration of the medicinal effects of Panax notoginseng, wild Panax notoginseng has disappeared due to uncontrolled excavation. Artificially cultivated Panax notoginseng has serious problems such as continuous cropping obstacles, diseases, heavy metal residues and long growth cycle, which greatly restrict the development of the industry.

[0003] With the gradual exploration of the medicinal effects of Panax notoginseng, wild Panax notoginseng has disappeared due to uncontrolled excavation. Artificially cultivated Panax notoginseng has serious problems such as continuous cropping obstacles, diseases, heavy metal residues and long growth cycle, which further restrict the supply of Panax notoginseng market. With the gradual expansion of Panax notoginseng product matrix to the fields of skin care and cosmetics, the requirements for raw material residues are becoming higher and higher, and it is difficult for ordinary field planting of Panax notoginseng to meet the requirements.

[0004] At present, the main method for producing Panax notoginseng adventitious roots is to use Panax notoginseng cotyledons, Panax notoginseng roots, Panax notoginseng young leaves, Panax notoginseng stem segments or Panax notoginseng callus to induce regenerated roots as explants, induce adventitious roots, and further expand through liquid culture in a triangular flask, which is not suitable for industrial production. Secondly, after inducing adventitious roots using cotyledons and other explants, the adventitious roots are cut into small pieces for culture, which has a long culture period and complex process, requires constant replacement of culture medium, has high cost, and limits its application in industrial production. The current Panax notoginseng adventitious root culture method has low proliferation rate and small biomass, which is difficult to meet the demand of industrial large-scale production. Conventional Panax notoginseng is diploid, and after doubling of its chromosomes, it has the characteristics of "large size of polyploidy" and large biomass. Therefore, it is necessary to provide a method for inducing Panax notoginseng tetraploid to improve the biomass and use a bioreactor to culture the Panax notoginseng tetraploid to improve the culture efficiency of Panax notoginseng tetraploid and obtain a large amount of Panax notoginseng adventitious roots. SUMMARY

[0005] To overcome the problems existing in the background technology, the present invention provides a method for inducing tetraploid Panax notoginseng to produce adventitious roots. By using chromosome doubling technology and bioreactor culture technology, a large number of Panax notoginseng adventitious roots are obtained, which improves the proliferation rate and biomass of Panax notoginseng. Furthermore, the combination of chromosome doubling technology and bioreactor can accelerate the growth cycle of Panax notoginseng adventitious roots, which greatly shortens the time from cultivation to harvest and improves production efficiency.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] This invention provides a method for inducing the generation of adventitious roots in tetraploid Panax notoginseng, comprising the following steps:

[0008] (1) Inducing embryogenic callus tissue using immature zygote of Panax notoginseng;

[0009] (2) Disperse the embryonic callus tissue into cells and sieve it through an 80-150 mesh sieve to obtain small-pore embryonic cell clusters. Place the small-pore embryonic cell clusters in an Erlenmeyer flask with 1-4 mg / mL colchicine and culture them in the dark.

[0010] (3) Remove colchicine to obtain cell clusters of uniform size, and place the cell clusters on a solid culture medium to induce tetraploid Panax notoginseng callus tissue;

[0011] (4) Tetraploid Panax notoginseng callus was placed on a plate culture medium for differentiation, and cotyledon embryos were obtained after 2-3 months;

[0012] (5) The cotyledon embryos were transferred to an intermittent submerged bioreactor for culture. After 1-2 months, a large number of adventitious roots grew from the cotyledon embryos in the intermittent submerged bioreactor.

[0013] In the above technical solution, the embryonic callus tissue described in step (2) is dispersed by stirring with a magnetic stirring rotor for 5-10 minutes.

[0014] In the above technical solution, the dark culture in step (2) is to place the Erlenmeyer flask on a shaker at 60 rpm and culture it in the dark for 24-72 hours.

[0015] In the above technical solution, the solid culture medium in step (3) is a WPM solid culture medium with 0.5-2 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 1 g / L activated carbon and 3 g / L Phytagel added.

[0016] In the above technical solution, the plate culture medium in step (4) is a WPM culture medium with 30 g / L sucrose, 0.5-1 g / L glutamine, 0.3-0.6% Phytagel, 1 g / L activated carbon, and pH 5.8.

[0017] In the technical solution, the step (5) intermittent submerged bioreactor is a 1L bioreactor.

[0018] In the technical solution, the step (5) intermittent submerged bioreactor is filled with 200-300 mL of WPM liquid medium added with NAA 0.25-0.5 mg / L, IBA 0.25-0.5 mg / L, 30 g / L sucrose, and pH is 5.8.

[0019] In the technical solution, the step (5) intermittent submerged bioreactor is filled with 200-300 mL of WPM liquid medium added with NAA 0.25-0.5 mg / L, IBA 0.25-0.5 mg / L, 30 g / L sucrose, and pH is 5.8.

[0020] Compared with the prior art, the beneficial effects of the present application are that:

[0021] 1. The present application uses chromosome doubling technology and bioreactor culture technology to obtain a large number of panax notoginseng adventitious roots, improves the proliferation rate and biomass of panax notoginseng, and the combination of chromosome doubling technology and bioreactor can accelerate the growth cycle of panax notoginseng adventitious roots, greatly shortens the time from culture to harvest, improves the production efficiency, has larger biomass, higher content of secondary metabolites, and larger rhizome.

[0022] 2. The panax notoginseng adventitious roots of the present application are directly produced from rhizome, which saves the step of induction from explants, has high growth efficiency, has high proliferation rate through intermittent submerged bioreactor culture, and the biomass is increased by 7.88 times within 4w of culture time.

[0023] 3. The present application uses bioreactor culture technology to provide a controllable and stable environment, and the culture conditions are not limited by natural conditions under artificial control, so that the culture of panax notoginseng adventitious roots can be carried out on a large scale, and uninterrupted production can be realized throughout the year, thereby meeting the large demand of the market for panax notoginseng medicinal materials, and the process is simple, the cost is low, the proliferation rate is high, the repeatability is good, and the growth rate of adventitious roots is fast.

[0024] 4. The present application can culture panax notoginseng without damaging natural resources and relying on land, breaks through the limitation of relying on land resources in the traditional planting process, does not need to damage the natural environment, ensures the green production of panax notoginseng medicinal materials, and has no chemical residues, so as to realize the pure and safe supply of raw materials; avoids the use of pesticides, effectively isolates the direct contact with the local soil containing heavy metals in Yunnan, fundamentally solves the problems of excessive pesticide residues and heavy metal pollution in traditional panax notoginseng planting, solves the long-existing problem of continuous cropping obstacles in traditional panax notoginseng planting, ensures the healthy growth of crops, and actively responds to the policy guidance of non-agricultural and non-food policy advocated by the state. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description only represent some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor based on the structures shown in the drawings.

[0026] Figure 1 is the effect picture of the present application for inducing adventitious roots of Panax notoginseng tetraploid at each stage;

[0027] Figure 2 is the effect picture of the present application for inducing adventitious roots;

[0028] Figure 3 is the effect picture of the present application for Panax notoginseng seedlings. DETAILED DESCRIPTION

[0029] In order to make the objects, technical solutions and advantages of the present application clearer, the following will further describe the embodiments of the present application in combination with the drawings.

[0030] The following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present application. Instead, they are merely examples of devices and methods consistent with some aspects of the present application as detailed in the appended claims.

[0031] In the description of the present application, it should be understood that the terms "first", "second" and the like are used only for the purpose of description and should not be understood as indicating or implying relative importance. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances. In addition, in the description of the present application, unless otherwise specified, "a plurality of" means two or more. "And / or", the association between the associated objects, means that there can be three kinds of relationships, for example, A and / or B, which can represent the three cases of A alone, A and B together, and B alone. The character " / " generally represents a "or" relationship between the associated objects before and after.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing the specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more associated listed items.

[0033] A method for inducing adventitious roots of Panax notoginseng tetraploid, comprising the following steps:

[0034] (1) Using immature zygotic embryos of Panax notoginseng to induce embryogenic callus;

[0035] (2) Dispersing the embryogenic callus by using a magnetic stirring rotor for 10 min, sieving by using a 80-150 mesh sieve, obtaining small-pore embryogenic cell clusters, and placing the small-pore embryogenic cell clusters in a triangular flask added with 1-4 mg / mL colchicine, and placing the triangular flask on a 60 rpm shaker for dark culture for 24-72 h;

[0036] (3) Removing the colchicine, obtaining cell clusters of uniform size, and placing the cell clusters on a WPM solid culture medium added with 0.5-2 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 1 g / L activated carbon and 3 g / L Phytagel to culture and induce tetraploid Panax notoginseng callus;

[0037] (4) Placing the tetraploid Panax notoginseng callus on a flat plate culture medium of WPM added with 30 g / L sucrose, 0.5-1 g / L glutamine, 0.3-0.6% Phytagel, 1 g / L activated carbon, and pH 5.8 to differentiate, and obtaining cotyledon embryos after 2-3 months;

[0038] (5) Transferring the cotyledon embryos into a 1L intermittent submerged bioreactor for culture, the intermittent submerged bioreactor is filled with 200-300 mL of WPM liquid culture medium added with NAA 0.25-0.5 mg / L, IBA 0.25-0.5 mg / L, 30 g / L sucrose, and pH 5.8, the immersion frequency is 4h / 2min, the inoculation amount is 0-3 g, and the light time is 16 / 8h, and a large amount of adventitious roots grow from the cotyledon embryos in the intermittent submerged bioreactor after 1-2 months.

[0039] Plant bioreactor technology is being widely applied in plant tissue culture process because of high degree of automation, cost saving, short production cycle and less influence from outside world, the change of chromosome number in a living organism is carried out in units of chromosome sets, when the chromosome number in a living organism reaches 3 sets or more than 3 sets, it is called polyploidy, compared with diploid plants, polyploid plants have unique advantages and potential commercial application value in many plants in terms of increased organ (larger and thicker leaves, thicker rhizomes) biomass and secondary metabolites. Combining the advantages of the two technologies can greatly shorten the production cycle and reduce the production cost.

[0040] Example 1

[0041] A method for inducing adventitious roots of Panax notoginseng tetraploid, comprising the following steps:

[0042] (1) inducing embryogenic callus from immature zygotic embryos of Panax notoginseng, using WPM woody plant medium with 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel (plant gel), and pH 5.8;

[0043] (2) placing the embryogenic callus in a sterile triangular flask containing 30 mL of culture solution using sterilized tweezers, placing a magnetic stirring rotor in the triangular flask, stirring for 10 min for cell dispersion, sieving using a 80-150 mesh sieve, obtaining small-pore embryogenic cell clusters, placing the small-pore embryogenic cell clusters in a triangular flask containing 1 mg / mL colchicine, and placing the triangular flask in a 60 rpm shaker for dark culture for 24 h;

[0044] (3) removing the colchicine, obtaining cell clusters of uniform size, and placing the cell clusters on WPM solid medium with 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 1 g / L activated carbon, and 3 g / L Phytagel to induce tetraploid Panax notoginseng callus;

[0045] (4) placing the tetraploid Panax notoginseng callus on a flat plate culture medium of WPM with 30 g / L sucrose, 1 g / L glutamine, 0.6% Phytagel, 1 g / L activated carbon, and pH 5.8 to differentiate, and obtaining cotyledon embryos after 2-3 months;

[0046] (5) transferring the cotyledon embryos into a 1L intermittent submerged bioreactor for culture, the intermittent submerged bioreactor containing 200 mL of WPM liquid medium with 30 g / L sucrose and pH 5.8, the immersion frequency being 4 h / 2 min, the inoculation amount being 0-3 g, and the light time being 16 / 8 h, and a large amount of adventitious roots growing from the cotyledon embryos in the intermittent submerged bioreactor after 1-2 months. The final determination shows that the inoculation amount is 0.538 g, the growth amount is 3.478 g, the proliferation coefficient is 6.46, and the number of roots is 3.25 ± 0.50.

[0047] Example 2

[0048] (1) inducing embryogenic callus from immature zygotic embryos of Panax notoginseng, using WPM woody plant medium with 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel (plant gel), and pH 5.8;

[0049] (2) In the clean bench, the embryogenic callus was picked up with sterilized tweezers and placed in a flask containing 30 mL of culture solution. A magnetic stirring rotor was placed in the flask, and the cells were dispersed by stirring for 10 min. The small-pore embryogenic cell clusters were obtained by sieving with a 80-150 mesh screen. The small-pore embryogenic cell clusters were placed in a flask containing 1 mg / mL colchicine, and the flask was placed on a shaker at 60 rpm for dark culture for 48 h;

[0050] (3) The colchicine was removed, and the cell clusters of uniform size were placed on a WPM solid medium containing 1 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 1 g / L activated carbon, and 3 g / L Phytagel to induce tetraploid Panax notoginseng callus;

[0051] (4) The tetraploid Panax notoginseng callus was placed on a flat plate medium of WPM containing 30 g / L sucrose, 0.6 g / L glutamine, 0.3% Phytagel, 1 g / L activated carbon, and pH 5.8 to differentiate, and cotyledon embryos were obtained after 2-3 months;

[0052] (5) The cotyledon embryos were transferred to a 1 L intermittent submerged bioreactor containing 300 mL of WPM liquid medium containing NAA 0.25 mg / L, 30 g / L sucrose, and pH 5.8. The immersion frequency was 4 h / 2 min, the inoculation amount was 0-3 g, and the light time was 16 / 8 h. After 1-2 months, a large number of adventitious roots grew from the cotyledon embryos in the intermittent submerged bioreactor. The final determination of the inoculation amount was 0.833 g, the growth amount was 5.394 g, the proliferation coefficient was 6.48, and the number of roots was 7.25 ± 3.50.

[0053] Example 3

[0054] A method for inducing tetraploid Panax notoginseng to produce adventitious roots, comprising the following steps:

[0055] (1) Embryogenic callus was induced using immature zygotic embryos of Panax notoginseng. The culture medium was WPM woody plant medium containing 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel (plant gel), and pH 5.8;

[0056] (2) In the sterile bench, the embryogenic callus was taken with sterilized tweezers and put into a flask containing 30 mL of culture solution. A magnetic stirring rotor was put into the flask. The cells were dispersed by stirring for 10 min. The small-pore embryogenic cell clusters were obtained by sieving with a 80-150 mesh screen. The small-pore embryogenic cell clusters were put into a flask containing 2 mg / mL colchicine. The flask was placed on a shaker at 60 rpm for dark culture for 48 h;

[0057] (3) The colchicine was removed, and the cell clusters of uniform size were obtained. The cell clusters were placed on a WPM solid medium containing 1.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 1 g / L activated carbon and 3 g / L Phytagel to induce tetraploid Panax notoginseng callus;

[0058] (4) The tetraploid Panax notoginseng callus was placed on a flat plate medium of WPM containing 30 g / L sucrose, 0.7 g / L glutamine, 0.5% Phytagel, 1 g / L activated carbon, and pH 5.8 to differentiate. Cotyledon embryos were obtained after 2-3 months;

[0059] (5) The cotyledon embryos were transferred into an intermittent immersion 1L bioreactor for culture. The intermittent immersion bioreactor contained 300 mL of WPM liquid medium containing NAA 0.5 mg / L and 30 g / L sucrose, pH 5.8. The immersion frequency was 4 h / 2 min, the inoculation amount was 0-3 g, and the light time was 16 / 8 h. A large number of adventitious roots were grown from the cotyledon embryos in the intermittent immersion bioreactor after 1-2 months. The final determination of the inoculation amount was 0.656 g, the growth amount was 5.036 g, the proliferation coefficient was 7.68, and the number of roots was 8.50 ± 3.11.

[0060] Example 4

[0061] A method for inducing tetraploid Panax notoginseng to produce adventitious roots, comprising the following steps:

[0062] (1) Embryogenic callus was induced using immature zygotic embryos of Panax notoginseng. The culture medium was WPM woody plant medium containing 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel (plant gel), and pH 5.8;

[0063] (2) In the clean bench, the embryogenic callus was taken with sterilized tweezers and placed in a flask containing 30 mL of culture solution. A magnetic stirring rotor was placed in the flask, and the cells were dispersed by stirring for 10 min. The small-pore embryogenic cell clusters were obtained by sieving with a 80-150 mesh screen. The small-pore embryogenic cell clusters were placed in a flask containing 3 mg / mL colchicine, and the flask was placed on a shaker at 60 rpm for dark culture for 60 h;

[0064] (3) The colchicine was removed, and the cell clusters of uniform size were placed on a WPM solid medium containing 1.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 1 g / L activated carbon, and 3 g / L Phytagel to induce tetraploid Panax notoginseng callus;

[0065] (4) The tetraploid Panax notoginseng callus was placed on a flat plate medium of WPM containing 30 g / L sucrose, 0.8 g / L glutamine, 0.6% Phytagel, 1 g / L activated carbon, and pH 5.8 to differentiate, and cotyledon embryos were obtained after 2-3 months;

[0066] (5) The cotyledon embryos were transferred to an intermittent immersion 1 L bioreactor for culture. The intermittent immersion bioreactor contained 300 mL of WPM liquid medium containing NAA 0.25 mg / L, IBA 0.25 mg / L, 30 g / L sucrose, and pH 5.8. The immersion frequency was 4 h / 2 min, the inoculation amount was 0-3 g, and the light time was 16 / 8 h. After 1-2 months, a large number of adventitious roots grew from the cotyledon embryos in the intermittent immersion bioreactor. The final determination of the inoculation amount was 0.914 g, the growth amount was 6.12 g, the proliferation coefficient was 6.70, and the number of roots was 5.75 ± 2.06.

[0067] Example 5

[0068] A method for inducing tetraploid Panax notoginseng to produce adventitious roots, comprising the following steps:

[0069] (1) Embryogenic callus was induced using immature zygotic embryos of Panax notoginseng. The culture medium was WPM woody plant medium containing 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel (plant gel), and pH 5.8;

[0070] (2) In the clean bench, the embryogenic callus was taken with sterilized tweezers and placed in a flask containing 30 mL of culture solution. A magnetic stirring rotor was placed in the flask, and the cells were dispersed by stirring for 10 min. The small-pore embryogenic cell clusters were obtained by sieving with a 80-150 mesh screen. The small-pore embryogenic cell clusters were placed in a flask containing 4 mg / mL colchicine, and the flask was placed on a shaker at 60 rpm for dark culture for 60 h;

[0071] (3) The colchicine was removed, and the cell clusters of uniform size were placed on a WPM solid medium containing 2 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 1 g / L activated carbon, and 3 g / L Phytagel to induce tetraploid Panax notoginseng callus;

[0072] (4) The tetraploid Panax notoginseng callus was placed on a flat plate medium of WPM containing 30 g / L sucrose, 0.8 g / L glutamine, 0.5% Phytagel, 1 g / L activated carbon, and pH 5.8 to differentiate, and cotyledon embryos were obtained after 2-3 months;

[0073] (5) The cotyledon embryos were transferred to an intermittent immersion 1 L bioreactor for culture. The intermittent immersion bioreactor contained 300 mL of WPM liquid medium containing NAA 0.25 mg / L, IBA 0.5 mg / L, 30 g / L sucrose, and pH 5.8. The immersion frequency was 4 h / 2 min, the inoculation amount was 0-3 g, and the light time was 16 / 8 h. After 1-2 months, a large number of adventitious roots grew from the cotyledon embryos in the intermittent immersion bioreactor. The final determination of the inoculation amount was 0.736 g, the growth amount was 4.935 g, the proliferation coefficient was 6.71, and the number of roots was 9.75 ± 5.56.

[0074] Example 6

[0075] A method for inducing tetraploid Panax notoginseng to produce adventitious roots, comprising the following steps:

[0076] (1) Embryogenic callus was induced using immature zygotic embryos of Panax notoginseng. The culture medium was WPM woody plant medium containing 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel (plant gel), and pH 5.8;

[0077] (2) In the sterile bench, the embryogenic callus was taken with sterilized tweezers and put into a flask containing 30 mL of culture solution. A magnetic stirring rotor was put into the flask. The cells were dispersed by stirring for 10 min. The small-pore embryogenic cell clusters were obtained by sieving with a 80-150 mesh screen. The small-pore embryogenic cell clusters were put into a flask containing 1.5 mg / mL colchicine. The flask was placed on a shaker at 60 rpm for dark culture for 72 h;

[0078] (3) The colchicine was removed, and the cell clusters of uniform size were obtained. The cell clusters were placed on a WPM solid medium containing 1.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 1 g / L activated carbon and 3 g / L Phytagel to induce tetraploid Panax notoginseng callus;

[0079] (4) The tetraploid Panax notoginseng callus was placed on a flat plate medium of WPM containing 30 g / L sucrose, 0.8 g / L glutamine, 0.5% Phytagel, 1 g / L activated carbon, and pH 5.8. After 2-3 months, cotyledon embryos were obtained;

[0080] (5) The cotyledon embryos were transferred into a 1 L intermittent submerged bioreactor for culture. The intermittent submerged bioreactor contained 300 mL of WPM liquid medium containing NAA 0.5 mg / L, IBA 0.25 mg / L, 30 g / L sucrose, and pH 5.8. The immersion frequency was 4 h / 2 min, the inoculation amount was 0-3 g, and the light time was 16 / 8 h. After 1-2 months, a large number of adventitious roots grew from the cotyledon embryos in the intermittent submerged bioreactor. The final determination showed that the inoculation amount was 0.727 g, the growth amount was 5.216 g, the proliferation coefficient was 67.17, and the number of roots was 11.50 ± 3.32.

[0081] Example 7

[0082] A method for inducing tetraploid Panax notoginseng to produce adventitious roots, comprising the following steps:

[0083] (1) Embryogenic callus was induced using immature zygotic embryos of Panax notoginseng. The culture medium was WPM woody plant medium containing 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel (plant gel), and pH 5.8;

[0084] (2) In the clean bench, the embryogenic callus was taken with sterilized tweezers and placed in a triangular flask containing 30 mL of culture solution. A magnetic stirring rotor was placed in the triangular flask. The cells were dispersed by stirring for 10 min. The small-pore embryogenic cell clusters were obtained by sieving with a 80-150 mesh screen. The small-pore embryogenic cell clusters were placed in a triangular flask containing 4 mg / mL colchicine. The triangular flask was placed on a shaker at 60 rpm for dark culture for 72 h;

[0085] (3) The colchicine was removed, and the cell clusters of uniform size were placed on a WPM solid culture medium containing 2 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 1 g / L activated carbon and 3 g / L Phytagel to induce tetraploid Panax notoginseng callus;

[0086] (4) The tetraploid Panax notoginseng callus was placed on a flat plate culture medium of WPM containing 30 g / L sucrose, 1 g / L glutamine, 0.6% Phytagel, 1 g / L activated carbon, and pH 5.8 to differentiate. Cotyledon embryos were obtained after 2-3 months;

[0087] (5) The cotyledon embryos were transferred into an intermittent immersion 1L bioreactor for culture. The intermittent immersion bioreactor contained 300 mL of WPM liquid culture medium containing NAA 0.5 mg / L, IBA 0.5 mg / L, 30 g / L sucrose, and pH 5.8. The immersion frequency was 4 h / 2 min, the inoculation amount was 0-3 g, and the illumination time was 16 / 8 h. A large number of adventitious roots were grown from the cotyledon embryos in the intermittent immersion bioreactor after 1-2 months. The final determination of the inoculation amount was 0.811 g, the growth amount was 6.389 g, the proliferation coefficient was 7.88, and the number of roots was 16.50 ± 3.87.

[0088] Experimental analysis

[0089] Table 1

[0090]

[0091] From the above data, it can be seen that the method of the present application has a significant effect on inducing tetraploid Panax notoginseng to produce adventitious roots. Compared with the control group (C), the treatment group added with NAA and IBA has a significant effect on promoting the growth of adventitious roots. The number of roots of the NAA 0.5 + IBA 0.5 treatment group reached 16.50 ± 3.87.

[0092]

[0093] Table 2

[0094] From the above data, it can be seen that when the colchicine concentration is 1.5 mg / mL and the treatment time is 72 h, the mixed ploidy induction rate is the highest, which is 14.29%.

[0095] Table 3

[0096]

[0097] The above data can show that the total saponin content of the tetraploid Panax notoginseng is doubled compared with that of the diploid Panax notoginseng.

[0098] The biomass of the adventitious roots is significantly improved through the optimized culture medium and the bioreactor technology, and the demand of large-scale production in industrialization is met; the process controllability is strong, the application of the bioreactor provides a stable and controllable culture environment, which is not limited by natural conditions, and the possibility of continuous production throughout the year is ensured; the method is ecological friendly, the dependence on land resources is avoided, the continuous cropping obstacles and heavy metal pollution problems in the traditional planting process are eliminated, and no pesticides are needed, so that the purity and safety of the adventitious roots are ensured.

[0099] Finally, it should be explained that the above preferred embodiments are only used to illustrate the technical solutions of the present application but not to limit the present application, and although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. A method for inducing adventitious root production in Panax notoginseng tetraploid, characterized by: The method for inducing the production of adventitious roots of the ginseng tetraploid comprises the following steps: (1) inducing embryogenic callus from immature zygotic embryos of ginseng; (2) dispersing the embryogenic callus, screening through a 80-150 mesh screen, obtaining small-pore embryogenic cell clusters, and placing the small-pore embryogenic cell clusters in a triangular flask with 2-4 mg / mL colchicine for dark culture for 48 h; (3) removing the colchicine, obtaining cell clusters of uniform size, and placing the cell clusters on a solid culture medium for culture to induce tetraploid ginseng callus; (4) placing the tetraploid ginseng callus on a plate culture medium for differentiation, and obtaining cotyledon embryos after 2-3 months; the plate culture medium is WPM medium with 30 g / L sucrose, 0.5-1 g / L glutamine, 0.3-0.6% Phytagel, 1 g / L activated carbon, and pH 5.8; (5) transferring the cotyledon embryos into a 1L intermittent submerged bioreactor for culture, the intermittent submerged bioreactor is filled with 200-300 mL of WPM liquid culture medium with 0.5 mg / L NAA, 0.5 mg / L IBA, 30 g / L sucrose, and pH 5.8; the immersion frequency of the cotyledon embryos transferred into the intermittent submerged bioreactor for culture is 4 h / 2 min, the inoculation amount is 3 g, and the illumination time is 16 / 8 h; a large amount of adventitious roots grow from the cotyledon embryos in the intermittent submerged bioreactor after 1-2 months.

2. The method of claim 1, wherein the method is characterized by: In step (2), the embryogenic callus is dispersed by stirring with a magnetic stirring rotor for 5-10 min.

3. The method of claim 1, wherein the method is characterized by: In step (2), the dark culture is performed by placing the triangular flask on a 60 rpm shaker for dark culture for 48 h.

4. The method of claim 1, wherein the method is characterized by: In step (3), the solid culture medium is WPM solid culture medium with 0.5-2 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 1 g / L activated carbon, and 3 g / L Phytagel.

Images