Method for inducing pseudo-ginseng tetraploid to generate adventitious roots

Abstract

The invention relates to a method for inducing pseudo-ginseng tetraploid to generate adventitious roots, which belongs to the technical field of biology and comprises the following steps: inducing embryogenic calluses by using immature zygotic embryos of pseudo-ginseng; performing cell dispersion on the embryogenic callus to obtain a small-aperture embryogenic cell cluster, and placing the small-aperture embryogenic cell cluster in a triangular flask added with 1-4mg / mL colchicine for dark culture; removing colchicine, placing the cell cluster on a solid culture medium, and culturing and inducing tetraploid pseudo-ginseng calluses; placing the tetraploid pseudo-ginseng callus on a plate culture medium for differentiation to obtain cotyledon embryos; and transferring the cotyledon embryo into an intermittent immersed bioreactor for culturing. A large number of pseudo-ginseng adventitious roots are obtained through the chromosome doubling technology and the bioreactor culture technology, the proliferation rate and biomass of pseudo-ginseng are improved, the growth cycle of the pseudo-ginseng adventitious roots can be accelerated through combination of the chromosome doubling technology and the bioreactor, the time from culture to harvesting is greatly shortened, and the production efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology, and particularly relates to a method for inducing adventitious roots in tetraploid Panax notoginseng. Background Art

[0002] Panax notoginseng, also known as Tianqi, belongs to the genus Panax of the Araliaceae family, together with ginseng, American ginseng, and Panax japonicus Torr., and is a perennial herb with a rhizome. Domestically, it is mainly distributed in Yunnan and Guangxi, growing under 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 the roots and rhizomes. "Supplementary Records of the Compendium of Materia Medica" records that "ginseng ranks first in invigorating qi, and Panax notoginseng ranks first in nourishing blood". Modern pharmacological research shows that total saponins of Panax notoginseng have functions such as anti - inflammation, regulating blood lipids, anti - platelet aggregation, anti - tumor, anti - thrombosis, preventing atherosclerosis and fatty liver, preventing myocardial infarction, promoting liver regeneration and angiogenesis. As the medicinal effects of Panax notoginseng are gradually discovered, wild Panax notoginseng has become extinct due to unrestrained excavation. Artificially cultivated Panax notoginseng has problems such as serious continuous cropping obstacles, diseases, heavy metal residues, and a long growth cycle, which greatly restricts the development of the industry.

[0003] As the medicinal effects of Panax notoginseng are gradually discovered, wild Panax notoginseng has become extinct due to over - exploitation. Artificially cultivated Panax notoginseng has problems such as serious continuous cropping obstacles, diseases, heavy metal residues, and a long growth cycle, which further restricts the supply of the Panax notoginseng market. And as the product matrix of Panax notoginseng gradually expands to fields such as skin care products and cosmetics, the requirements for raw material residues are getting higher and higher, and it is difficult for Panax notoginseng grown in ordinary fields to meet the requirements.

[0004] Currently, for the production of adventitious roots of Panax notoginseng, the main method is: using the roots of regenerated seedlings induced from the cotyledons, roots, young leaves, stem segments of Panax notoginseng or the callus of Panax notoginseng as explants to induce adventitious roots, and then further expanding through liquid culture in Erlenmeyer flasks, which is not suitable for industrial production. Secondly, after using cotyledons, etc. as explants to induce adventitious roots, the adventitious roots are cut into small segments for culture. The culture cycle is long, and due to the complexity of the process, the culture medium needs to be replaced continuously, resulting in high costs, which limits its application in industrial production. Currently, the proliferation rate of the adventitious root culture method of Panax notoginseng is low, and the biomass is small, making it difficult to meet the requirements of large - scale industrial production. Conventionally cultivated Panax notoginseng is diploid, and after its chromosome doubling, it has the characteristic of large biomass brought by "the gigantism of polyploids". Therefore, it is necessary to provide a method for inducing tetraploid Panax notoginseng to increase the biomass and use a bioreactor to improve the culture efficiency of tetraploid Panax notoginseng to obtain a large amount of adventitious roots of Panax notoginseng. Summary of the Invention

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

[0006] To achieve the above object, the present invention is realized through the following technical solutions:

[0007] The present invention provides a method for inducing adventitious roots in Panax notoginseng tetraploids, comprising the following steps:

[0008] (1) Inducing embryogenic callus from immature zygotic embryos of Panax notoginseng;

[0009] (2) Dispersing the embryogenic callus cells, sieving through a 80 - 150 mesh sieve to obtain embryogenic cell clusters with small pore diameters, and placing the embryogenic cell clusters with small pore diameters in an Erlenmeyer flask containing 1 - 4 mg / mL colchicine for dark culture;

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

[0011] (4) Differentiating the tetraploid Panax notoginseng callus on a plate medium, and obtaining cotyledon embryos after 2 - 3 months;

[0012] (5) Transferring the cotyledon embryos into an intermittent immersion bioreactor for culture, and after 1 - 2 months, a large number of adventitious roots grow from the cotyledon embryos in the intermittent immersion bioreactor.

[0013] In the above technical solution, in step (2), the embryogenic callus is stirred with a magnetic stirring rotor for 5 - 10 min for cell dispersion.

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

[0015] In the above technical solution, in step (3), the solid medium is a WPM solid medium supplemented 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.

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

[0017] In the above technical solution, the intermittent immersion bioreactor described in step (5) is a 1L bioreactor.

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

[0019] In the above technical solution, the immersion frequency of transferring cotyledon embryos into the intermittent immersion bioreactor for cultivation in step (5) is 4 h / 2 min, the inoculation amount is 0 - 3 g, and the light duration is 16 / 8 h.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1. The present invention uses chromosome doubling technology and bioreactor cultivation technology to obtain a large number of Panax notoginseng adventitious roots, improving the proliferation rate and biomass of Panax notoginseng. Moreover, the combination of chromosome doubling technology and the bioreactor can accelerate the growth cycle of Panax notoginseng adventitious roots, greatly shortening the time from cultivation to harvest and improving production efficiency. The biomass is larger, the content of secondary metabolites is higher, and the rhizome is larger.

[0022] 2. The adventitious roots of Panax notoginseng in the present invention are directly generated from the rhizome, eliminating the step of inducing from explants, with high growth efficiency. Through cultivation in an intermittent immersion bioreactor, the proliferation rate is high, and within 4 weeks of cultivation, the biomass increases by 7.88 times.

[0023] 3. The present invention uses bioreactor cultivation technology to provide a controllable and stable environment. The cultivation conditions are not restricted by natural conditions under artificial control, enabling the large-scale cultivation of Panax notoginseng adventitious roots, and enabling year-round continuous production, thus meeting the large market demand for Panax notoginseng medicinal materials. Moreover, 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 invention can cultivate Panax notoginseng without destroying natural resources and without relying on land, breaking through the limitation of relying on land resources in the traditional planting process, without the need to damage the natural environment, ensuring the green production of Panax notoginseng medicinal materials without any chemical residues, thus realizing a pure and safe raw material supply; avoiding the use of pesticides and effectively isolating direct contact with heavy metal-containing soil in Yunnan. This not only fundamentally solves the problems of excessive pesticide residues and heavy metal pollution in traditional Panax notoginseng planting, but also solves the long-existing continuous cropping obstacle problem in traditional Panax notoginseng planting, ensuring the healthy growth of crops. Description of the Drawings

[0025] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0026] Figure 1 are the effect diagrams of each stage of inducing adventitious roots in tetraploid Panax notoginseng of the present invention;

[0027] Figure 2 is the effect diagram of producing adventitious roots of the present invention;

[0028] Figure 3 is the effect diagram of Panax notoginseng seedlings of the present invention. Detailed implementation manners

[0029] To make the objectives, technical solutions and advantages of the present invention clearer, the following will further describe in detail the embodiments of the present application in conjunction with the drawings.

[0030] When the following description involves the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementation manners described in the following exemplary embodiments do not represent all the implementation manners consistent with the present invention. On the contrary, they are only examples of the devices and methods consistent with some aspects of the present invention as detailed in the appended claims.

[0031] In the description of the present invention, it should be understood that the terms "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. In addition, in the description of the present invention, unless otherwise specified, "a plurality of" means two or more. "And / or" describes the association relationship of associated objects and indicates that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. The character " / " generally represents an "or" relationship between the front and rear associated objects.

[0032] Unless otherwise defined, all the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

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

[0034] (1) Induce embryogenic callus from immature zygotic embryos of Panax notoginseng;

[0035] (2) Stir the embryogenic callus with a magnetic stirrer rotor for 10 min for cell dispersion, sieve it through a sieve with 80 - 150 meshes to obtain embryogenic cell clusters with small pore diameters, place the embryogenic cell clusters with small pore diameters in a triangular flask containing 1 - 4 mg / mL colchicine, and place the triangular flask on a shaker at 60 rpm for dark culture for 24 h - 72 h;

[0036] (3) Remove colchicine to obtain cell clusters of uniform size, place the cell clusters on a WPM solid medium supplemented 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 for culture to induce tetraploid Panax notoginseng callus;

[0037] (4) Place the tetraploid Panax notoginseng callus on a WPM plate medium supplemented 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 for differentiation, and obtain cotyledon embryos after 2 - 3 months;

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

[0039] Plant bioreactor technology is being widely used in plant tissue culture due to its advantages such as high automation, cost savings, short production cycle, and less influence from the outside world. The change in the number of chromosomes in an organism occurs in units of chromosome sets. When the number of chromosomes in an organism reaches 3 sets or more, it is called polyploid. Compared with diploid plants, polyploid plants have unique advantages and potential commercial application values in traits such as enlarged organs (bigger and thicker leaves, thicker roots and stems), biomass, and secondary metabolites. Combining the advantages of these two technologies can greatly shorten the production cycle and reduce production costs.

[0040] Example 1

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

[0042] (1) Induce embryogenic callus from immature zygotic embryos of Panax notoginseng. The medium is WPM woody plant medium supplemented with 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel, and with a pH of 5.8.

[0043] (2) In a laminar flow hood, use sterilized forceps to pick up the embryogenic callus and place it into a triangular flask containing 30 mL of culture solution. A magnetic stirring rotor is placed in the triangular flask, and stir for 10 min to disperse the cells. Then, filter through a sieve with a mesh size of 80 - 150 meshes to obtain embryogenic cell clusters with small pore diameters. Place the embryogenic cell clusters with small pore diameters into a triangular flask containing 1 mg / mL colchicine, and place the triangular flask on a shaker at 60 rpm for dark culture for 24 h.

[0044] (3) Remove the colchicine to obtain cell clusters of uniform size. Place the cell clusters on a WPM solid medium supplemented 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 for culture to induce tetraploid Panax notoginseng callus.

[0045] (4) Place the tetraploid Panax notoginseng callus on a plate medium of WPM supplemented with 30 g / L sucrose, 1 g / L glutamine, 0.6% Phytagel, 1 g / L activated carbon, and with a pH of 5.8 for differentiation. After 2 - 3 months, cotyledon embryos are obtained.

[0046] (5) Transfer the cotyledon embryos to a 1 L intermittent immersion bioreactor for culture. The intermittent immersion bioreactor is filled with 200 mL of WPM liquid medium supplemented with 30 g / L sucrose and with a pH of 5.8. The immersion frequency is 4 h / 2 min, the inoculation amount is 0 - 3 g, the light time is 16 / 8 h. After 1 - 2 months, a large number of adventitious roots grow from the cotyledon embryos in the intermittent immersion bioreactor. Finally, it is determined 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) Induce embryogenic callus from immature zygotic embryos of Panax notoginseng. The medium is WPM woody plant medium supplemented with 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel, and with a pH of 5.8.

[0049] (2) Use sterilized forceps to pick up embryogenic callus in a laminar flow hood and place it into a triangular flask containing 30 mL of culture medium. Put a magnetic stirring rotor into the triangular flask, stir for 10 min for cell dispersion, sieve through a sieve with 80 - 150 meshes to obtain embryogenic cell clusters with small pore diameters. Place the embryogenic cell clusters with small pore diameters into a triangular flask containing 1 mg / mL colchicine, and place the triangular flask on a shaker at 60 rpm for dark culture for 48 h;

[0050] (3) Remove colchicine to obtain cell clusters with uniform size. Place the cell clusters on a WPM solid medium supplemented with 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 for culturing to induce tetraploid Panax notoginseng callus;

[0051] (4) Place the tetraploid Panax notoginseng callus on a WPM plate medium supplemented with 30 g / L sucrose, 0.6 g / L glutamine, 0.3% Phytagel, 1 g / L activated carbon, and with a pH of 5.8 for differentiation. Cotyledon embryos can be obtained after 2 - 3 months;

[0052] (5) Transfer the cotyledon embryos into a 1 L temporary immersion bioreactor for culturing. The temporary immersion bioreactor is filled with 300 mL of WPM liquid medium supplemented with 0.25 mg / L NAA, 30 g / L sucrose, and with a pH of 5.8. The immersion frequency is 4 h / 2 min, the inoculation amount is 0 - 3 g, the light time is 16 / 8 h. After 1 - 2 months, a large number of adventitious roots grow from the cotyledon embryos in the temporary immersion bioreactor. Finally, the measured inoculation amount is 0.833 g, the growth amount is 5.394 g, the proliferation coefficient is 6.48, and the number of roots is 7.25 ± 3.50.

[0053] Example 3

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

[0055] (1) Induce embryogenic callus from immature zygotic embryos of Panax notoginseng, and the culture medium is WPM woody plant medium supplemented with 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel (phytagel), and with a pH of 5.8;

[0056] (2) Use sterilized forceps to pick up embryogenic callus in a laminar flow hood and place it into a triangular flask containing 30 mL of culture medium. Put a magnetic stirring rotor into the triangular flask, stir for 10 min for cell dispersion, sieve through a sieve with 80 - 150 meshes to obtain embryogenic cell clusters with small pore diameters. Place the embryogenic cell clusters with small pore diameters into a triangular flask containing 2 mg / mL colchicine, and place the triangular flask on a shaker at 60 rpm for dark culture for 48 h;

[0057] (3) Remove colchicine to obtain cell clusters of uniform size, and place the cell clusters on a WPM solid medium supplemented with 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 for culturing to induce tetraploid Panax notoginseng callus;

[0058] (4) Place the tetraploid Panax notoginseng callus on a WPM plate medium supplemented with 30 g / L sucrose, 0.7 g / L glutamine, 0.5% Phytagel, 1 g / L activated carbon, and with a pH of 5.8 for differentiation, and cotyledon embryos can be obtained after 2 - 3 months;

[0059] (5) Transfer the cotyledon embryos to a 1 L intermittent immersion bioreactor for culturing. The intermittent immersion bioreactor is filled with 300 mL of WPM liquid medium supplemented with 0.5 mg / L NAA, 30 g / L sucrose, and with a pH of 5.8. The immersion frequency is 4 h / 2 min, the inoculation amount is 0 - 3 g, the light time is 16 / 8 h. After 1 - 2 months, a large number of adventitious roots grow from the cotyledon embryos in the intermittent immersion bioreactor. Finally, the measured inoculation amount is 0.656 g, the growth amount is 5.036 g, the proliferation coefficient is 7.68, and the number of roots is 8.50 ± 3.11.

[0060] Example 4

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

[0062] (1) Induce embryogenic callus from immature zygotic embryos of Panax notoginseng, and the culture medium is a WPM woody plant medium supplemented with 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel (phytagel), and with a pH of 5.8;

[0063] (2) In a laminar flow hood, use sterilized forceps to pick up the embryogenic callus and put it into a triangular flask containing 30 mL of culture solution. Place a magnetic stirring rotor in the triangular flask, stir for 10 min to disperse the cells, sieve through an 80 - 150 mesh sieve to obtain embryogenic cell clusters with small pore diameters, and place the embryogenic cell clusters with small pore diameters in a triangular flask supplemented with 3 mg / mL colchicine. Place the triangular flask on a shaker at 60 rpm for dark culture for 60 h;

[0064] (3) Remove colchicine to obtain cell clusters of uniform size, and place the cell clusters on a WPM solid medium supplemented with 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 for culturing to induce tetraploid Panax notoginseng callus;

[0065] (4) Place the tetraploid Panax notoginseng callus on a flat medium of WPM supplemented with 30 g / L sucrose, 0.8 g / L glutamine, 0.6% Phytagel, 1 g / L activated carbon, and a pH of 5.8 for differentiation. After 2 - 3 months, cotyledon embryos are obtained.

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

[0067] Example 5

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

[0069] (1) Induce embryogenic callus from immature zygotic embryos of Panax notoginseng. The culture medium is WPM woody plant medium supplemented with 0.5 mg / L 2,4 - D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel (phytogel), and a pH of 5.8.

[0070] (2) In a laminar flow hood, use sterilized forceps to pick up the embryogenic callus and place it in a triangular flask containing 30 mL of culture solution. Put a magnetic stirring rotor in the triangular flask and stir for 10 min to disperse the cells. Sieve through an 80 - 150 mesh sieve to obtain embryogenic cell clusters with small pore diameters. Place the embryogenic cell clusters with small pore diameters in a triangular flask containing 4 mg / mL colchicine, and place the triangular flask on a shaker at 60 rpm for dark culture for 60 h.

[0071] (3) Remove the colchicine to obtain cell clusters of uniform size. Place the cell clusters on a solid medium of WPM supplemented with 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 for culturing to induce tetraploid Panax notoginseng callus.

[0072] (4) Place the tetraploid Panax notoginseng callus on a flat medium of WPM supplemented with 30 g / L sucrose, 0.8 g / L glutamine, 0.5% Phytagel, 1 g / L activated carbon, and a pH of 5.8 for differentiation. After 2 - 3 months, cotyledon embryos are obtained.

[0073] (5) Transfer the cotyledon embryos into a 1L intermittent immersion bioreactor for cultivation. The intermittent immersion bioreactor is filled with 300 mL of WPM liquid medium supplemented with 0.25 mg / L NAA, 0.5 mg / L IBA, 30 g / L sucrose, and with a pH of 5.8. The immersion frequency is 4 h / 2 min, the inoculation amount is 0 - 3 g, the light time is 16 / 8 h. After 1 - 2 months, a large number of adventitious roots grow from the cotyledon embryos in the intermittent immersion bioreactor. Finally, the inoculation amount is determined to be 0.736 g, the growth amount is 4.935 g, the proliferation coefficient is 6.71, and the number of roots is 9.75 ± 5.56.

[0074] Example 6

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

[0076] (1) Induce embryogenic callus from immature zygotic embryos of Panax notoginseng. The culture medium is WPM woody plant medium supplemented with 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel, and with a pH of 5.8;

[0077] (2) In a laminar flow hood, use sterilized forceps to pick up the embryogenic callus and put it into a triangular flask containing 30 mL of culture solution. Place a magnetic stirring rotor in the triangular flask and stir for 10 min to disperse the cells. Sieve through a sieve with 80 - 150 meshes to obtain embryogenic cell clusters with small pore sizes. Place the embryogenic cell clusters with small pore sizes in a triangular flask containing 1.5 mg / mL colchicine, and place the triangular flask on a shaker at 60 rpm for dark culture for 72 h;

[0078] (3) Remove the colchicine to obtain cell clusters of uniform size. Place the cell clusters on a WPM solid medium supplemented with 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 for culturing to induce tetraploid Panax notoginseng callus;

[0079] (4) Place the tetraploid Panax notoginseng callus on a plate medium of WPM supplemented with 30 g / L sucrose, 0.8 g / L glutamine, 0.5% Phytagel, 1 g / L activated carbon, and with a pH of 5.8 for differentiation. After 2 - 3 months, cotyledon embryos are obtained;

[0080] (5) Transfer the cotyledon embryos into a 1 L intermittent immersion bioreactor for cultivation. The intermittent immersion bioreactor is filled with 300 mL of WPM liquid medium supplemented with 0.5 mg / L NAA, 0.25 mg / L IBA, 30 g / L sucrose, and with a pH of 5.8. The immersion frequency is 4 h / 2 min, the inoculation amount is 0 - 3 g, the light time is 16 / 8 h. After 1 - 2 months, a large number of adventitious roots grow from the cotyledon embryos in the intermittent immersion bioreactor. Finally, the measured inoculation amount is 0.727 g, the growth amount is 5.216 g, the proliferation coefficient is 67.17, and the number of rooted plants is 11.50 ± 3.32.

[0081] Example 7

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

[0083] (1) Induce embryogenic callus from immature zygotic embryos of Panax notoginseng. The medium is WPM woody plant medium supplemented with 0.5 mg / L 2,4-D, 1 g / L PVP, 1 g / L CH, 30 g / L sucrose, 3 g / L Phytagel, and with a pH of 5.8;

[0084] (2) In a laminar flow hood, use sterilized forceps to pick up the embryogenic callus and put it into a triangular flask containing 30 mL of culture solution. Place a magnetic stirring rotor in the triangular flask and stir for 10 min to disperse the cells. Sieve through an 80 - 150 mesh sieve to obtain embryogenic cell clusters with small pore diameters. Place the embryogenic cell clusters with small pore diameters in a triangular flask supplemented with 4 mg / mL colchicine, and place the triangular flask on a shaker at 60 rpm for dark culture for 72 h;

[0085] (3) Remove the colchicine to obtain cell clusters of uniform size. Place the cell clusters on a WPM solid medium supplemented with 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 for cultivation to induce tetraploid Panax notoginseng callus;

[0086] (4) Place the tetraploid Panax notoginseng callus on a plate medium of WPM supplemented with 30 g / L sucrose, 1 g / L glutamine, 0.6% Phytagel, 1 g / L activated carbon, and with a pH of 5.8 for differentiation. After 2 - 3 months, cotyledon embryos are obtained;

[0087] (5) The cotyledon embryos were transferred to a 1L intermittent submerged bioreactor for culture. The intermittent submerged bioreactor was filled with 300mL of WPM liquid medium with 0.5mg / L NAA, 0.5mg / L IBA, 30g / L sucrose, pH 5.8, immersion frequency 4h / 2min, inoculation amount 0-3g, light duration 16 / 8h, and after 1-2 months, the cotyledon embryos grew a large number of adventitious roots in the intermittent submerged bioreactor. The final inoculation amount was 0.811g, the growth amount was 46.389g, 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 invention shows a significant effect in inducing the production of adventitious roots in tetraploid Panax notoginseng. Compared with the control group (C), the treatment group with the addition of NAA and IBA has a significant effect in promoting the growth of adventitious roots, among which the number of roots in the NAA 0.5+IBA 0.5 treatment group reached 16.50±3.87.

[0092] Table 2

[0093]

[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] From the above data, it can be seen that the total saponin content of tetraploid Panax notoginseng is doubled compared with that of diploid Panax notoginseng.

[0098] The present invention significantly increases the biomass. Through the optimized culture medium and bioreactor technology, the biomass of adventitious roots is significantly increased, meeting the needs of industrial large-scale production; the process controllability is strong, and the application of the bioreactor provides a stable and controllable culture environment, which is not restricted by natural conditions and ensures the possibility of continuous production throughout the year; the method is eco-friendly and avoids dependence on land resources, eliminates the continuous cropping obstacles and heavy metal pollution problems in the traditional planting process, and does not require the use of pesticides, ensuring the purity and safety of the adventitious roots.

[0099] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for inducing tetraploid Panax notoginseng to produce adventitious roots, characterized in that: The method for inducing Panax notoginseng tetraploid to produce adventitious roots specifically comprises the following steps: (1) Inducing embryonic callus from immature zygotic embryos of Panax notoginseng; (2) dispersing the embryonic callus, sieving it through a 80-150 mesh sieve to obtain small-pore embryonic cell clusters, and placing the small-pore embryonic cell clusters in a conical flask added with 1-4 mg / mL colchicine for dark culture; (3) removing colchicine to obtain cell clusters of uniform size, and culturing the cell clusters on solid culture medium to induce tetraploid Panax notoginseng callus; (4) Placing tetraploid Panax notoginseng callus on a plate culture medium for differentiation, and obtaining cotyledon embryos after 2-3 months; (5) The cotyledon embryos are transferred to an intermittent submerged bioreactor for culture. After 1-2 months, the cotyledon embryos grow a large number of adventitious roots in the intermittent submerged bioreactor.

2. The method for inducing tetraploid Panax notoginseng to produce adventitious roots according to claim 1, characterized in that: In step (2), the embryonic callus tissue is stirred for 5-10 minutes using a magnetic stirring rotor to disperse the cells.

3. The method for inducing tetraploid Panax notoginseng to produce adventitious roots according to claim 1, characterized in that: The dark culture in step (2) is to place the flask on a shaker at 60 rpm for 24-72 hours.

4. The method for inducing tetraploid Panax notoginseng to produce adventitious roots according to claim 1, characterized in that: The solid culture medium in step (3) is a WPM solid culture medium supplemented 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.

5. The method for inducing tetraploid Panax notoginseng to produce adventitious roots according to claim 1, characterized in that: The plate culture medium in step (4) is a WPM culture medium supplemented with 30 g / L sucrose, 0.5-1 g / L glutamine, 0.3-0.6% Phytagel, 1 g / L activated carbon, and a pH of 5.

8.

6. The method for inducing tetraploid Panax notoginseng to produce adventitious roots according to claim 1, characterized in that: The intermittent submerged bioreactor in step (5) is a 1L bioreactor.

7. The method for inducing tetraploid Panax notoginseng to produce adventitious roots according to claim 6, characterized in that: The intermittent submerged bioreactor in step (5) is filled with 200-300 mL of WPM liquid culture medium with 0.25-0.5 mg / L NAA, 0.25-0.5 mg / L IBA, and 30 g / L sucrose added and a pH of 5.

8.

8. The method for inducing tetraploid Panax notoginseng to produce adventitious roots according to any one of claims 1, 6 or 7, characterized in that: In step (5), the cotyledon embryos are transferred to an intermittent submerged bioreactor for culture with an immersion frequency of 4 h / 2 min, an inoculation amount of 0-3 g, and a lighting time of 16 / 8 h.

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