Induction method, regeneration method and genetic transformation method of schisandra chinensis embryogenic callus

Abstract

The invention discloses an induction method, a regeneration method and a genetic transformation method of schisandra chinensis embryogenic callus, and relates to the field of transgenosis, the induction method comprises the following steps: taking any one or more of new shoot meristem, subterranean stem tip, petiole, petal, corolla and pistil of schisandra chinensis as explants for embryogenic callus induction; the selection of explants of the schisandra chinensis regeneration system is enriched, and the method has important significance for breaking seasonal limitation of sampling of the schisandra chinensis regeneration system. In addition, a genetic transformation method of schisandra chinensis is established, and a new way is provided for obtaining schisandra chinensis transgenic plants.

Description

Technical Field

[0001] The invention relates to the field of transgenics, and in particular to an induction method, a regeneration method and a genetic transformation method of Schisandra chinensis embryonic callus. Background Art

[0002] At present, somatic embryogenesis is widely used to establish plant regeneration system because of its advantages of strong proliferation ability, high genetic stability and good structural integrity. In the existing research, Schisandra chinensis mainly uses dormant buds as explants to induce embryonic callus, which has certain seasonal limitations.

[0003] At present, traditional genetic transformation methods, such as leaf disc method, gene gun method, electric shock method, ultrasonic method, microinjection method, pollen tube channel method, PEG (polyethylene glycol) method, etc., face problems such as low rooting rate, low transformation efficiency and genetic instability. However, the Agrobacterium-mediated genetic transformation pathway based on somatic embryogenesis has high genetic stability. Embryonic cells are in close contact with Agrobacterium during the infection process, which can achieve a high genetic transformation success rate and also avoid chimeras produced by other pathways. Agrobacterium-mediated genetic transformation technology is in a leading position in the field of transgenics due to its wide application, clear mechanism and mature methods. At present, there is no relevant report on the genetic transformation of Schisandra chinensis in domestic and foreign research.

[0004] In the genetic transformation process with embryonic callus as the receptor, there is a phenomenon that the embryonic callus is easy to scatter in the liquid and difficult to pick up. After infection, the high concentration of antibiotics used in the inhibition process of Agrobacterium will have a toxic effect on the plant, while low concentrations are difficult to inhibit.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide an induction method, a regeneration method and a genetic transformation method for Schisandra chinensis embryonic callus.

[0007] The present invention is achieved in that:

[0008] In the first aspect, an embodiment of the present invention provides a method for inducing embryonic callus of Schisandra chinensis, which comprises: inoculating Schisandra chinensis explants into an embryonic callus induction medium for culturing to obtain induced embryonic callus; selecting embryonic callus with good properties from the induced embryonic callus and inoculating them into an embryonic callus proliferation medium for culturing; wherein the Schisandra chinensis explants comprise: any one or more of shoot meristem, underground stem apex, petiole, petal, corolla, and pistil.

[0009] In a second aspect, an embodiment of the present invention provides a method for regenerating Schisandra chinensis plants, comprising: obtaining embryonic callus using the induction method described in the above embodiment.

[0010] In a third aspect, an embodiment of the present invention provides a method for genetic transformation of Schisandra chinensis, comprising: using the embryonic callus obtained by the induction method described in the above embodiment as an explant for genetic transformation.

[0011] The present invention has the following beneficial effects:

[0012] The induction method includes inducing embryonic callus tissue by using any one or more of the new shoot meristem, underground stem tip, petiole, petal, corolla and pistil of Schisandra chinensis as explants, enriching the selection of explants for Schisandra chinensis regeneration system, and having important significance for breaking the seasonal limitation of sampling of Schisandra chinensis regeneration system. In addition, the invention establishes a genetic transformation method of Schisandra chinensis, providing a new way to obtain Schisandra chinensis transgenic plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0014] Figure 1 The effect of carbon nanotubes on the proliferation of embryonic callus; A to D are the proliferation of embryonic callus in liquid culture medium with 0, 10, 20, and 30 mg / L carbon nanotubes added, respectively;

[0015] Figure 2 is the inhibitory effect of Timentin on Agrobacterium; A1 to A6 are the proliferation of Agrobacterium in the presence of 0, 100, 200, 300, 400, and 500 mg / L Timentin, respectively;

[0016] Figure 3 eGFP fluorescence measurement of embryonic callus and somatic embryos; A1 is wild-type callus under normal light conditions, B1 is transgenic callus under normal light conditions, C1 is wild-type somatic embryos under normal light conditions, D1 and E1 are transgenic somatic embryos under normal light conditions. A2 is wild-type callus under blue fluorescence, B2 is transgenic callus under blue fluorescence, C2 is wild-type somatic embryos under blue fluorescence, D2 and E2 are transgenic somatic embryos under blue fluorescence, and F is transgenic somatic embryos and wild-type somatic embryos under the same blue fluorescence;

[0017] Figure 4 are the results of DNA extraction and PCR identification; A is the result of DNA extraction; B is the result of PCR identification; maker is 1900 bp, 1, 2, 3 are transgenic embryonic calli, 4, 5, 6 are transgenic somatic embryos, 7, 8, 9 are transgenic plants, 10 is wild-type embryonic callus, 11 is wild-type somatic embryos, 12 is wild-type plants, and 13 is pCAMBIA1301 plasmid;

[0018] Figure 5 This is the process of plant regeneration after infection of Schisandra chinensis embryonic callus. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0020] The embodiment of the present invention constructs a set of new methods for constructing Schisandra chinensis regeneration system and genetic transformation system, which fills the research gap of Schisandra chinensis regeneration system and genetic transformation system. Compared with the prior art, dormant buds are used as explants, and underground stem tips, stem tip meristems, petioles, petals, corollas, and pistils are used as explants to induce embryonic callus, enriching the selection of explants in the Schisandra chinensis regeneration system, which is of great significance for breaking the seasonal restrictions on sampling of the Schisandra chinensis regeneration system. The embodiment of the present invention also finds and verifies that the addition of carbon nanotubes (CNTs) is conducive to the induced proliferation of Schisandra chinensis embryonic callus, and verifies the beneficial effects of adding nutrients such as hydrolyzed casein (CH), inositol (GC) and glutamine (L-Gln) on Schisandra chinensis embryonic callus in the process of somatic embryo differentiation. The method has high construction efficiency, strong repeatability, and is simple and easy to implement.

[0021] On the one hand, an embodiment of the present invention provides a method for inducing embryonic callus of Schisandra chinensis, which comprises: inoculating Schisandra chinensis explants into an embryonic callus induction medium for culturing to obtain induced embryonic callus; selecting embryonic callus with good properties from the induced embryonic callus and inoculating them into an embryonic callus proliferation medium for culturing; wherein the Schisandra chinensis explants comprise: any one or more of shoot meristem, underground stem apex, petiole, petal, corolla, and pistil.

[0022] In some embodiments, the outer diameter of the Schisandra chinensis explant is 1-10 mm, and the outer diameter can be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mm or a range between any two thereof.

[0023] In some embodiments, the callus induction medium is supplemented with hydrolyzed casein.

[0024] In some embodiments, the final concentration of hydrolyzed casein in the callus induction medium is 0.1-1 g / L, and the concentration can specifically be any one of 0.1, 0.2, 0.4, 0.6, 0.8, 1 g / L or a range between any two of them.

[0025] In some embodiments, the components included in the callus induction medium and the final concentrations of the components are as follows: MS medium, 1-5 mg / L 2,4-D, 20-40 g / L sucrose, 5-8 g / L agar, and 0.1-1 g / L hydrolyzed casein. Specifically, the final concentration of 2,4-D can be any one of 1, 2, 2.5, 3, 3.5, 4, and 5 mg / L or a range between any two. The final concentration of sucrose can be any one of 20, 25, 30, 35, and 40 g / L or a range between any two. The final concentration of agar can be any one of 5, 5.5, 6, 6.5, 7, 7.5, and 8 g / L or a range between any two. The final concentration of hydrolyzed casein can be any one of 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, and 1 g / L or a range between any two.

[0026] In some embodiments, the culturing step in the embryonic callus induction medium includes: culturing in the dark for 1 to 5 days, then culturing under light, and the culturing conditions under light include: a culturing temperature of 20 to 30° C., a lighting time of 12 to 20 h, and a light intensity of 80 to 160 μmol m -2 s -1 Specifically, the culture temperature can be any one of 20, 22, 24, 25, 26, 28, 30°C or a range between any two thereof. The illumination time can be any one of 12, 14, 16, 18, 20h or a range between any two thereof. The illumination intensity can be 80, 100, 120, 140, 160 μmol m -2 s -1 Any one or a range between any two of the .

[0027] In some embodiments, the embryogenic callus proliferation medium is a liquid medium.

[0028] In some embodiments, carbon nanotubes (CNTs) are added to the embryonic callus proliferation medium. In the embodiments of the present invention, liquid culture medium is used to add CNTs to proliferate embryonic callus, which increases the proliferation rate of embryonic callus and is beneficial to improving the regeneration efficiency of Schisandra chinensis.

[0029] In some embodiments, the final concentration of the carbon nanotubes in the embryonic callus proliferation medium is 10-40 mg / L, specifically any one of 10, 15, 20, 25, 30, 35, 40 mg / L or a range between any two of them.

[0030] In some embodiments, the components included in the embryonic callus proliferation medium and the final concentrations of the components are as follows: MS medium, 0.1-3 mg / L 2,4-D, 20-40 g / L sucrose, 0.1-1 g / L hydrolyzed casein and 10-40 mg / L carbon nanotubes. Specifically, the final concentration of 2,4-D can be any one of 0.1, 0.5, 1, 1.5, 2, 2.5, 3 mg / L or a range between any two. The final concentration of sucrose can be any one of 20, 25, 30, 35, 40 g / L or a range between any two. The final concentration of hydrolyzed casein can be any one of 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, 1 g / L or a range between any two. The final concentration of carbon nanotubes can be any one of 10, 15, 20, 25, 30, 35, 40 mg / L or a range between any two.

[0031] In some embodiments, the culture conditions in the embryonic callus proliferation medium include: a culture temperature of 20 to 30° C., 140 to 180 r / min, a light exposure time of 12 to 20 h, and a light intensity of 80 to 160 μmol m -2 s -1 Specifically, the culture temperature can be any one of 20, 22, 24, 25, 26, 28, 30°C or a range between any two thereof. The culture speed can be any one of 140, 150, 160, 170, 180 r / min or a range between any two thereof. The illumination time can be any one of 12, 14, 16, 18, 20 h or a range between any two thereof. The illumination intensity can be 80, 100, 120, 140, 160 μmol m -2 s -1 Any one or a range between any two of the .

[0032] In some embodiments, the good traits include embryonic callus with milky white color and / or obvious granular structure.

[0033] On the other hand, an embodiment of the present invention provides a method for regenerating Schisandra chinensis plants, comprising: obtaining embryonic callus using the induction method described in any of the aforementioned embodiments.

[0034] In some embodiments, the regeneration method further comprises: transferring the embryonic callus to a somatic embryo induction medium for culturing to obtain somatic embryos.

[0035] In some embodiments, the regeneration method further comprises washing the embryonic callus before transferring to the somatic embryo induction medium. The washing can be carried out with water. The water can be sterile water.

[0036] In some embodiments, the somatic embryo induction medium is a solid medium.

[0037] In some embodiments, the somatic embryo induction medium is supplemented with any one or more of sucrose, agar and exogenous nutrients. The exogenous nutrients include any one or more of glutamine, hydrolyzed casein and inositol. The addition of exogenous nutrients is beneficial to improving the regeneration efficiency of Schisandra chinensis embryonic callus differentiation into somatic embryos.

[0038] In some embodiments, the final concentration of sucrose in the somatic embryo induction medium is 10-30 g / L, specifically any one of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 g / L or a range between any two thereof. In some embodiments, the final concentration of agar in the somatic embryo induction medium is 5-8 g / L, specifically any one of 5, 5.5, 6, 6.5, 7, 7.5, 8 g / L or a range between any two thereof. In some embodiments, the final concentration of exogenous nutrients in the somatic embryo induction medium is 500-1000 mg / L, specifically any one of 500, 600, 700, 800, 900, 1000 mg / L or a range between any two thereof. The concentration of exogenous nutrients defined in the embodiments of the present invention is conducive to significantly promoting the proliferation and development of Schisandra chinensis embryonic cells.

[0039] In some embodiments, the culture conditions in the somatic embryo induction medium include: 20-30°C, dark culture for 50-70 days. Specifically, the culture temperature can be any one of 20, 22, 24, 25, 26, 28, 30°C or a range between any two. The light intensity can be 100, 110, 120, 130, 140 μmol m -2 s -1 The culture time can be any one of 50, 55, 60, 65, 70 days or a range between any two of them.

[0040] In some embodiments, the regeneration method further comprises: transferring the somatic embryos to be cultured under light conditions, wherein the light intensity is 100 to 140 μmol m -2 s-1 , until they differentiate into regenerated plants.

[0041] In some embodiments, the regeneration method further comprises transplanting the regenerated plants.

[0042] In addition, an embodiment of the present invention further provides a method for genetic transformation of Schisandra chinensis, comprising: using the embryonic callus obtained by the induction method described in any of the above embodiments as an explant to perform genetic transformation.

[0043] In some embodiments, the genetic transformation comprises the following steps: infecting the explant with an Agrobacterium infection solution containing the target gene.

[0044] In some embodiments, the infection time is 15 to 30 minutes, specifically any one of 15, 16, 18, 20, 22, 24, 26, 28, 30 minutes or a range between any two of them.

[0045] In some embodiments, the OD of the infection fluid is 600 The value is 0.4 to 0.8, and specifically can be any one of 0.4, 0.5, 0.6, 0.7, 0.8 or a range between any two of them.

[0046] In some embodiments, before the infection, the genetic transformation method further comprises: pre-culturing the explants before infection.

[0047] In some embodiments, the pre-culture medium used in the pre-culture includes the following components and the final concentrations of the components: MS medium, 20-40 g / L sucrose, 0.1-3 mg / L 2,4-D and 20-40 mg / L carbon nanotubes. Specifically, the final concentration of sucrose can be any one of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 g / L or a range between any two. The final concentration of 2,4-D can be any one of 0.1, 0.5, 1, 1.5, 2, 3 mg / L or a range between any two. The final concentration of carbon nanotubes can be any one of 20, 25, 30, 35, 40 mg / L or a range between any two.

[0048] The pre-culture step can maintain the activity of embryonic callus, make it have good regeneration ability, improve the survival rate and regeneration efficiency of embryonic callus after infection, and improve the infection efficiency. The carbon nanotubes added in the pre-culture process can absorb the secondary metabolites produced during the growth and development of embryonic callus, play an antioxidant role, maintain the embryonic ability of embryonic callus and somatic embryos, and improve the proliferation and differentiation efficiency.

[0049] In some embodiments, the pre-culture conditions include: 20-30°C, 100-140 r / min, and a light intensity of 140-160 μmol m -2 s -1 , 1 to 5 days. Specifically, the culture temperature can be any one of 20, 22, 24, 25, 26, 28, 30°C or a range between any two thereof. The rotation speed can be any one of 140, 150, 160, 170, 180 r / min or a range between any two thereof. The light intensity can be 100, 120, 140 μmol m -2 s -1 The pre-culture time can be any one of 1, 2, 3, 4, 5 days or a range between any two of them.

[0050] In some embodiments, the infected explants are transferred to a co-cultivation medium for co-cultivation.

[0051] In some embodiments, acetosyringone (As) is added to the co-cultivation medium.

[0052] In some embodiments, the co-culture medium includes the following components and their final concentrations: MS medium, 20-40 g / L sucrose, 0.1-3 mg / L 2,4-D, 0.1-10 mg / L carbon nanotubes, and 50-300 μmol / L acetosyringone.

[0053] In some embodiments, the final concentration of acetosyringone in the co-cultivation medium is 50 to 300 μmol / L, specifically any one of 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300 μmol / L or a range between any two. This concentration range is beneficial to improving the transformation efficiency of Agrobacterium. The final concentration of sucrose can be any one of 20, 25, 30, 35, 40 g / L or a range between any two. The final concentration of 2,4-D can be any one of 0.1, 0.5, 1, 2, 2.5, 3 mg / L or a range between any two. The final concentration of carbon nanotubes is any one of 0.1, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / L or a range between any two.

[0054] In some embodiments, the co-culture period is 1 to 4 days, specifically any one of 1, 2, 3, 4 days or a range between any two of them.

[0055] In some embodiments, after co-cultivation, the genetic transformation method further comprises: performing any one or more steps of eluting, screening and differentiating the product after co-cultivation.

[0056] In some embodiments, the elution comprises eluting the co-cultured product in an elution solution; the elution solution comprises an elution medium and / or water (specifically sterile water).

[0057] In some embodiments, the components included in the elution medium and the final concentrations of the components are as follows: 1 / 3M medium, 10-30 g / L sucrose, 0.5-0.9 g / L inositol and 0.1-0.5 g / L Timentin. Specifically, the final concentration of sucrose can be any one of 20, 25, 30, 35, 40 g / L or a range between any two. The final concentration of inositol can be any one of 0.5, 0.6, 0.7, 0.8, 0.9 g / L or a range between any two. The final concentration of Timentin can be any one of 0.1, 0.2, 0.3, 0.4, 0.5 g / L or a range between any two.

[0058] The elution medium can effectively elute Agrobacterium, inhibit the growth of Agrobacterium, and reduce the damage of Agrobacterium to explants, thereby obtaining sterile transgenic plants.

[0059] In some embodiments, the number of elutions using the elution medium is 1 to 4 times, specifically any one of 1, 2, 3, 4 times or a range between any two thereof. The number of elutions using water is 1 to 4 times, specifically any one of 1, 2, 3, 4 times or a range between any two thereof.

[0060] In some embodiments, after each elution using the elution solution, the elution further comprises filtering using a cell sieve to filter out the eluted product.

[0061] The embodiment of the present invention adds an elution step, reduces the residual Agrobacterium, and gradually reduces the dosage of antibiotics, thereby improving the survival rate of embryonic callus or somatic embryos while inhibiting bacteria.

[0062] In some embodiments, the screening adopts screening culture medium for screening culture to screen out resistant tissues, and the screening culture medium includes components and the final concentrations of the components are: 1 / 3M culture medium, 20-40g / L sucrose, 5-8g / L agar, 0.5-1g / L glutamine, 0.1-0.5g / L kanamycin (kana) and 0.1-0.5g / L timentin. Specifically, the final concentration of sucrose can be any one of 20, 25, 30, 35, 40g / L or a range between any two. The final concentration of agar can be any one of 5, 5.5, 6, 6.5, 7, 7.5, 8g / L or a range between any two. The final concentration of glutamine can be any one of 0.5, 0.6, 0.7, 0.8, 0.9, 1g / L or a range between any two. The final concentration of kanamycin can be any one of 0.5, 0.6, 0.7, 0.8, 0.9, 1 g / L or a range between any two thereof. The final concentration of timentin can be any one of 0.1, 0.2, 0.3, 0.4, 0.5 g / L or a range between any two thereof.

[0063] In some embodiments, the screening culture conditions include: 20-30° C., dark culture, 12-16 days. Specifically, the culture temperature can be any one of 20, 22, 24, 25, 26, 28, 30° C. or a range between any two thereof. The culture time can be any one of 12, 13, 14, 15, 16 days or a range between any two thereof.

[0064] In some embodiments, the differentiation comprises: placing the screened product in a first differentiation medium for differentiation culture, and then transferring it to a second differentiation medium for differentiation culture until it differentiates into a plant.

[0065] The first differentiation medium comprises: 1 / 3M medium, 20-40 g / L sucrose, 5-8 g / L agar and 0.5-1 g / L glutamine. Specifically, the final concentration of sucrose can be any one of 20, 25, 30, 35, 40 g / L or a range between any two. The final concentration of agar can be any one of 5, 5.5, 6, 6.5, 7, 7.5, 8 g / L or a range between any two. The final concentration of glutamine can be any one of 0.5, 0.6, 0.7, 0.8, 0.9, 1 g / L or a range between any two.

[0066] The second differentiation medium includes components and their final concentrations are as follows: MS medium, 20-40 g / L sucrose and 5-8 g / L agar. Specifically, the final concentration of sucrose can be any one of 20, 25, 30, 35, 40 g / L or a range between any two. The final concentration of agar can be any one of 5, 5.5, 6, 6.5, 7, 7.5, 8 g / L or a range between any two.

[0067] In some embodiments, the culture conditions when using the first differentiation medium include: 20-30° C., dark culture, 50-70 days. Specifically, the culture temperature can be any one of 20, 22, 24, 25, 26, 28, 30° C. or a range between any two of them. The culture time can be any one of 50, 55, 60, 65, 70 days or a range between any two of them.

[0068] In some embodiments, the culture conditions when using the second differentiation medium include: 20-30° C., culture under light. The culture temperature can be any one of 20, 22, 24, 25, 26, 28, 30° C. or a range between any two of them. The culture under light refers to normal natural light conditions without any special restrictions.

[0069] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0070] The specific formula of the culture medium used in the subsequent examples is:

[0071] Example 1

[0072] This embodiment provides a method for regenerating Schisandra chinensis plants, which specifically includes the following steps.

[0073] (1) Obtaining sterile explants

[0074] Trial selection: In this example, the "Zaohong" germplasm resource Schisandra chinensis cultivated in the Schisandra chinensis germplasm resource garden of Jilin Agricultural University in Changchun, Jilin Province, China was used to collect new shoot meristems.

[0075] In April and May, we randomly selected 'Zaohong' Schisandrae Chinensis, which was growing well and healthy, and collected the new shoot meristem as experimental materials.

[0076] Wash the explants with sterile water by shaking for 3 to 4 times to remove dust on the surface; shake with 75% alcohol for 30 seconds and pour out the alcohol; rinse with sterile water for 3 times; pour 0.1% mercuric chloride solution into the bottle on the clean bench, set the shaking disinfection time to 10 minutes, and pour out the mercuric chloride; finally, rinse with sterile water for 3 times and place in a 4°C refrigerator for later use.

[0077] (2) Embryogenic callus induction

[0078] Place the sterilized explants in a culture dish, and cut off the new shoot meristem (2 mm) under sterile conditions using tweezers and a blade on a clean bench; inoculate the explants into triangular bottles containing culture medium, 3 in each bottle; place the tissue culture bottles in the tissue culture room, and after three days of dark culture, transfer to light for culture and observation.

[0079] The formula of callus induction medium is: MS+2,4-D 3mg / L+sucrose 30g / L+agar 6.5g / L+CH0.5g / L;

[0080] The culture conditions under light were: 25 ± 1 °C, light duration of 16 h, light intensity of 120 μmol m -2 s -1 .

[0081] (3) Proliferation of embryonic callus

[0082] From the induced embryonic callus, the ones with milky white color and obvious granular structure were picked and transferred to the embryonic callus proliferation medium. MS liquid medium was used and CNTs were added. The final concentration of CNTs in the medium was 20 mg / L. 4 g of embryonic callus was inoculated in each bottle of culture medium. 10 bottles were inoculated for each treatment, and the test results were statistically analyzed after one month.

[0083] The formula of callus proliferation medium is: MS+2,4-D 1mg / L+sucrose 30g / L+CH 0.5g / L+CNTs 20mg / L;

[0084] The culture conditions were: 25 ± 1 °C, 160 r / min, photoperiod of 16 h, and light intensity of 120 μmol m -2 s -1 .

[0085] (4) Somatic embryo induction and plant regeneration

[0086] The proliferating embryonic callus was screened out in the liquid culture medium using a cell sieve, the remaining CNTs were repeatedly rinsed off with sterile water, and the callus was transferred to the somatic embryo induction medium. Each bottle of culture medium was inoculated with 4 g of embryonic callus, 10 bottles were inoculated, and the results were repeated three months later.

[0087] The formula of somatic embryo induction medium is: 1 / 3MS+sucrose 20g / L+agar 6.5g / L+L-Gln 750mg / L.

[0088] The culture conditions were: 25 ± 1 °C, dark culture for 2 months, then transferred to light conditions, light intensity 120 μmol m - 2 s -1, subculture every 30 days until differentiation into regenerated plants.

[0089] (5) Transplantation

[0090] Select regenerated plants with more roots, longer roots and stronger roots; loosen the cap of the tissue culture bottle and culture them in the tissue culture room for 5 days; open the cap and culture for 3 to 4 days before taking out the tissue culture seedlings; discard the culture medium and gently rinse the roots of Schisandra chinensis with tap water; bury them in the sterilized soil matrix while ensuring that the soil is moist; put the transplanted seedlings in a greenhouse, water them once a week and observe the growth of the plants.

[0091] Example 2

[0092] Effects of different explants on the induction rate of embryonic callus.

[0093] Six experimental groups were set up based on the regeneration method of Example 1, one group was the same as Example 1, and the remaining five groups were different from Example 1 mainly in that the explants were different. The six groups of experiments respectively selected new shoot meristem, underground stem apex, petiole, petal, corolla, and pistil. The 1% mercuric chloride oscillation disinfection time in step (1) was set to 14 minutes for underground stem apex, 10 minutes for new shoot meristem and petiole, and 5 minutes for petals, corolla, and pistil. The size of the explants cut in step (2) was set to new shoot meristem (2 mm), underground stem apex (5 mm), petiole (4 mm), petal (5 mm), corolla (5 mm), and pistil (5 mm).

[0094] The results are as follows.

[0095] Table 1 Effects of different explants on embryonic callus induction

[0096] deal with explant Embryogenic callus induction rate Time to induce callus (days) 1 New shoot meristem <![CDATA[34.44±1.92 a ]]> 30 2 Underground stem tip <![CDATA[4.44±1.92 e ]]> 60 3 Petiole <![CDATA[11.11±1.92 d ]]> 45 4 petal <![CDATA[25.56±3.85 b ]]> 36 5 corolla <![CDATA[16.67±3.33 c ]]> 40 6 pistil <![CDATA[22.22±1.92 b ]]> 32

[0097] Explant type plays an important role in callus induction. In the present embodiment, 6 explants all successfully induced embryonic callus, and the induction time was 30 days for the new shoot meristem, 60 days for the underground stem apex, 45 days for the petiole, 36 days for the petal, 40 days for the corolla, and 32 days for the pistil. The embryonic callus induction rate of the underground stem apex was the lowest, which was 4.4%, while the embryonic callus induction rate of the new shoot meristem was the highest, which was 34.44%, followed by the petal, which was 25.56%. Combined with the induction efficiency and required time of embryonic callus, the new shoot meristem was selected as the best explant for inducing embryonic callus. The influence of embodiment 3 carbon nanotubes on the proliferation of embryonic callus.

[0098] Three control groups were set up based on the regeneration method of Example 1. The regeneration method of the control group was substantially the same as that of Example 1, except that the amount of CNTs added to the callus proliferation medium in step (3) was different, namely 10, 30 and 40 mg / L.

[0099] Effects of carbon nanotubes on embryonic callus using shoot meristem as explants Figure 1 and Table 2.

[0100] Table 2 Effects of carbon nanotubes on the proliferation of embryonic callus

[0101]

[0102]

[0103] Depend on Figure 1 As shown in Table 2, the proliferation rate of embryonic callus treated with different concentrations of CNTs was significantly higher than that of the control group, indicating that adding CNTs to the suspension culture of embryonic callus can promote the proliferation of embryonic callus, and with the increase of concentration, it shows a trend of first increasing and then decreasing. The proliferation rate of embryonic callus reached the highest when the concentration was 20 mg / L, which is the optimal concentration for adding carbon nanotubes.

[0104] Example 4

[0105] Effects of different exogenous nutrients on somatic embryo differentiation.

[0106] Based on the regeneration method of Example 1, 9 groups of experiments were set up, of which 1 group was the same as Example 1, and the regeneration methods of the remaining 8 groups were roughly the same as Example 1, except that the nutrients in the somatic embryo induction medium in step (4) were different. The embryonic callus induction medium of each experimental group was as follows.

[0107] 1 / 3MS+sucrose 20g / L+agar 6.5g / L+GC (1, 3, 5) g / L;

[0108] 1 / 3MS+sucrose 20g / L+agar 6.5g / L+L-Gln (500, 750, 1000) mg / L;

[0109] 1 / 3MS+sucrose 20g / L+agar 6.5g / L+CH(500, 750, 1000)mg / L.

[0110] The results of the effects of different exogenous nutrients on embryonic callus with shoot meristem as explants are shown in Table 3.

[0111] Table 3 Effects of different exogenous nutrients on somatic embryo differentiation

[0112]

[0113] The results show that compared with the control group, different concentrations of GC, L-Gln, and CH significantly increased the proliferation rate of Schisandra chinensis somatic embryos. 0.75g and 1g of L-glutamine can significantly promote the differentiation of somatic embryos. Combined with the proliferation rate and somatic embryo differentiation, adding 0.75g L-glutamine is the best treatment.

[0114] Example 5

[0115] This embodiment provides a method for genetic transformation of Schisandra chinensis, which comprises the following steps.

[0116] (1) Screening of Kanamycin Selection Pressure

[0117] The embryonic callus obtained in step (3) of Example 1 and the somatic embryos of step (4) were used as explants and inoculated into a culture medium containing different kanamycin concentration gradients. The concentration gradient of kanamycin was set to 50, 100, 150, 200, and 250 mg / L. Ten bottles were inoculated for each treatment, and 4 embryonic callus or somatic embryos were inoculated in each bottle, with 1 g of each embryonic callus or somatic embryo, and the growth of embryonic callus and somatic embryos was observed and counted.

[0118] The formula of kanamycin screening medium is: MS + sucrose 30g / L + 2,4-D 1.0mg / L + CNTs 20mg / L + kana (50, 100, 150, 200, 250) mg / L;

[0119] The culture conditions were: 25 ± 1 °C, 160 r / min, photoperiod of 16 h, and light intensity of 120 μmol m -2 s -1 .

[0120] The results of embryonic callus and somatic embryos using shoot meristem as explants are shown in Table 4.

[0121] Table 4 Effects of different concentrations of kanamycin on the proliferation of embryonic callus and somatic embryos

[0122] serial number Kana concentration (mg / L) Fresh weight of embryonic callus (g) Embryogenic callus proliferation rate (%) Somatic embryo fresh weight (g) Somatic embryo proliferation rate (%) 1 0 <![CDATA[7.58±0.27 a ]]> <![CDATA[252.56±8.9 a ]]> <![CDATA[6.97±0.54 a ]]> <![CDATA[173.4±0.67 a ]]> 2 50 <![CDATA[7.34±0.08 b ]]> <![CDATA[244.67±2.65 b ]]> <![CDATA[6.31±0.5 b ]]> <![CDATA[157.67±1.23 b ]]> 3 100 <![CDATA[6.87±0.12 c ]]> 228.89±3.83c <![CDATA[5.79±0.06 c ]]> <![CDATA[144.83±1.42 c ]]> 4 150 <![CDATA[5.13±0.09 d ]]> <![CDATA[171.11±2.91 d ]]> <![CDATA[4.97±0.06 d ]]> <![CDATA[124.25±1.39 c ]]> 5 200 <![CDATA[4.30±0.03 e ]]> <![CDATA[143.44±1.02 e ]]> <![CDATA[4.37±0.4 e ]]> <![CDATA[109.33±1.01 e ]]> 6 250 <![CDATA[4.22±0.04 e ]]> <![CDATA[140.78±1.39 e ]]> <![CDATA[4.30±0.04 e ]]> <![CDATA[107.42±1.04 e ]]>

[0123] 200mg / L of Kana basically inhibited the proliferation of embryonic callus and somatic embryos of Schisandra chinensis, and can be used for screening positive plants.

[0124] (2) Screening of antibiotic concentration

[0125] The proliferated embryonic callus obtained in step (3) of Example 1 and the somatic embryos of step (4) were used as explants and inoculated on a culture medium containing different timentin concentration gradients. The timentin concentration gradient was set to 0, 100, 200, 300, 400, and 500 mg / L. Ten bottles were inoculated for each treatment, and 4 pieces of embryonic callus or somatic embryos were inoculated in each bottle, with 1 g of each embryonic callus or somatic embryo. After one month, the growth of embryonic callus and somatic embryos was observed and counted.

[0126] 50 μL of Bacillus GV3101 was added to 50 mL of LB liquid culture medium, and different levels of timentin (0, 100, 200, 300, 400 and 500 mg) were added, and the culture was shaken (200 r / min) at 28°C for 48 h to observe the growth inhibition of Agrobacterium by timentin.

[0127] The results of embryonic callus using shoot meristem as explants are shown in Figure 2 and Table 5.

[0128] Table 5 Effects of different concentrations of Timentin on the proliferation of embryonic callus and somatic embryos

[0129]

[0130] As the concentration of Timentin increased, the growth of Agrobacterium was inhibited. Figure 2 , A4) completely inhibited the growth of Agrobacterium, while 200 mg / L of Timentin ( Figure 2 However, a concentration of 400 mg / L of Timentin did not affect the growth of normal embryonic callus and did not produce serious toxicity to embryonic callus. A concentration of 500 mg / L of Timentin significantly inhibited the proliferation of embryonic callus (Table 5). Therefore, 300 mg / L of Timentin was selected to inhibit the growth of Agrobacterium.

[0131] (3) Pre-culture treatment

[0132] Embryogenic callus was inoculated into pre-culture medium and cultured at 25°C, 160 r / min, 120 μmol m -2 s -1 Pre-culture was performed under light conditions for 3 days;

[0133] The formula of the pre-culture medium is: MS+sucrose 30g / L+2,4-D 1.0mg / L+CNTs 30mg / L.

[0134] (4) Agrobacterium activation and infection fluid preparation

[0135] Take out the Agrobacterium strain frozen in a -80℃ refrigerator, take 2μL of the plant expression vector 1301 (empty vector) plasmid stored in the laboratory and 100μL of Agrobacterium competent cells EHA105, put them into a 1.5ml sterile centrifuge tube and mix them; ice bath for 30min; heat shock at 42℃ for 90s in a constant temperature water bath; quickly ice bath for 2min after taking out; add 900μL of antibiotic-free YEB liquid culture medium and mix them thoroughly; pour the bacterial solution into a 15ml sterile centrifuge tube, put it in a shaker, activate it at 140r / min for 1d, the temperature is 25℃, and shake the bacterial solution until it becomes obviously turbid; in the clean bench, use a sterile pipette tip to dip an appropriate amount of bacterial solution and apply it on a plate containing kanamycin and rifampicin. On a flat YEB solid culture medium; draw lines on the culture dish in a Z shape, as far away from the edge of the culture medium as possible; seal the culture dish, turn it upside down and culture it at 28°C for 3-4 days; when obvious single colonies grow on the culture dish, pick up an appropriate amount of colonies with a sterile pipette tip on the clean bench and transfer them into 40ml YEB culture medium containing kanamycin and rifampicin; after sealing, place it in a 130r / min shaker at 28°C for 16h overnight; pour the cultured turbid bacterial solution into a centrifuge tube for centrifugation; discard the supernatant, pour a certain volume of resuspension into the centrifuge tube (the volume is determined by the OD value required by the experimental design), mix it gently with a pipette, and finally take a small amount of resuspension to measure it with a spectrophotometer, and adjust the OD 600 The value is 0.6, and the preparation of the infection solution is completed; the prepared infection solution needs to be activated in a shaking incubator at 28°C and 120r / min for 2 to 3 hours; the embryonic callus tissue is placed in a centrifuge tube containing the infection solution, sealed, and placed in a shaking incubator at 28°C and 130r / min for 25 minutes (infection time) and taken out.

[0136] (5) Co-culture

[0137] The infected embryonic callus was inoculated into the co-cultivation medium and cultured at 25°C, 160 r / min, in the dark for one day (culture conditions).

[0138] The formula of co-culture medium: MS+sucrose 30 g / L+2,4-D 1.0 mg / L+CNTs 2.0 mg / L+As 200 μmol / L.

[0139] (6) Elution

[0140] ① On the clean bench, scoop the co-cultivated embryonic callus into the elution medium with a sterilized long spoon; ② Elute at 140r / min for 10 minutes and let stand for 2 minutes; ③ Filter out the embryonic callus with a sterilized cell sieve; ④ Use a sterilized long spoon to scoop the embryonic callus into a triangular flask containing sterile water; ⑤ Elute at 140r / min for 5 minutes and let stand for 2 minutes; ⑥ Filter out the embryonic callus with a sterilized cell sieve; ⑦ Use a sterilized long spoon to scoop into the elution medium; ⑧ Repeat steps ⑤ and ⑥ three times; ⑨ Use sterilized filter paper to absorb the remaining water.

[0141] The elution medium formula is:

[0142] 1 / 3MS+sucrose 20g / L+GC 700mg / L+Timentin 300mg / L.

[0143] (7) Screening and differentiation

[0144] The eluted embryonic callus was transferred to the screening medium, cultured at 25±1°C in the dark for 14 days, then transferred to the first differentiation medium, cultured at 25±1°C in the dark for two months, and then transferred to the second differentiation medium, cultured under light until it differentiated into regenerated plants;

[0145] The formula of screening medium is: 1 / 3MS+sucrose 30g / L+agar 6.5g / L+L-Gln 750mg / L+kana 200mg / L+Timentin 300mg / L;

[0146] The formula of the first differentiation medium is: 1 / 3MS + sucrose 30g / L + agar 6.5g / L + L-Gln 750mg / L;

[0147] The formula of the second differentiation medium is: MS+sucrose 30 g / L+agar 6.5 g / L.

[0148] (8) Identification of positive plants

[0149] The plant expression vector carries the eGFP fluorescent tag, and a handheld lamp is used to study the expression and fluorescence signal of eGFP in the resistant transgenic callus and somatic embryos, and the wild type is used as a control to further screen positive plants. DNA is extracted from the screened positive plants for PCR identification.

[0150] Results Figure 3 and Figure 4 .

[0151] pCAMBIA1301 carries the eGFP gene, which shows a fluorescent response under a blue excitation light source. Under fluorescence, the resistant transgenic embryonic callus ( Figure 3 , B2) and somatic embryos ( Figure 3, D2, E2) showed clear orange-yellow fluorescence; in contrast, the untransformed embryogenic callus ( Figure 3 , A2) showed no fluorescence response, while non-transformed somatic embryos ( Figure 3 , C2) only showed red fluorescence stimulated by chloroplasts. The fluorescence response of untransformed somatic embryos and successfully transformed somatic embryos under the same intensity excitation light source was significantly different ( Figure 3 , E). The results showed that the expression of eGFP in embryonic callus and somatic embryos was stable. Under the optimal transformation conditions, the embryonic callus transformation mediated by Agrobacterium tumefaciens EHA105 had a good effect.

[0152] Transgenic plants were identified by PCR, and DNA of transgenic embryonic callus, transgenic somatic embryos, transgenic plants, and wild-type embryonic callus, wild-type somatic embryos, and wild-type plants was detected. Figure 4 As shown, with the plasmid as the positive control, all transgenic embryonic calli, transgenic somatic embryos, and transgenic plants showing eGFP fluorescence showed an expansion of the expected size, confirming the presence of the transgene, while the wild type did not show an expansion of the expected size.

[0153] Example 6

[0154] Effects of key factors on genetic transformation efficiency.

[0155] Based on the regeneration method of Example 4, 16 groups of experiments were set up. The regeneration method of the experimental group was roughly the same as that of Example 4, except that the infection time of step (4) was different and the OD of the infection solution before infection in step (4) was 600 The values ​​are different, the number of co-cultivation days in step (5) is different, and the As added concentration in the co-cultivation medium in step (5) is different, see Table 6.

[0156] Table 6 Parameter settings of key factors

[0157]

[0158]

[0159] The results are shown in Table 7.

[0160] Table 7 Effects of key factors on genetic transformation efficiency

[0161]

[0162] The results show that the bacterial solution concentration, infection time, co-culture time and acetosyringone concentration of Agrobacterium have a significant effect on the transformation efficiency. When the bacterial solution concentration is too high, Agrobacterium grows too fast, and subsequent sterilization is difficult, resulting in the death of embryonic callus. With the increase of infection time, the expression rate of GUS gene in embryonic callus increases, but too long infection time may cause Agrobacterium to be too toxic to embryonic callus, and Agrobacterium is difficult to eliminate, resulting in the death of embryonic callus. When the infection time is too short, the embryonic callus cannot be effectively infected. The length of co-culture time has a greater impact on the transformation efficiency, because the transfer of T-DNA in plant cells can occur at this stage. Too long co-culture time will lead to excessive growth of Agrobacterium, Agrobacterium is not easy to clean, and the state of the recipient material is poor after sterilization. Acetosyringone is widely used to improve the efficiency of plant genetic transformation, but too high a concentration will have a certain toxicity to embryonic callus. Combining the above four factors, the optimal transformation condition is OD 600 =0.6, infection for 25 min, co-culture for 1 day, acetosyringone concentration was 200 μmol / L, at which time the transformation efficiency reached the maximum value of 32.43%.

[0163] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for inducing embryonic callus of Schisandra chinensis, characterized in that: It includes: Inoculating Schisandra chinensis explants into an embryonic callus induction medium for culturing to obtain induced embryonic callus; Selecting the embryonic callus with good properties from the induced embryonic callus and inoculating it into an embryonic callus proliferation medium for culturing; Wherein, the Schisandra chinensis explant comprises any one or more of new shoot meristem, underground stem tip, petiole, petal, corolla and pistil.

2. The induction method according to claim 1, characterized in that: The embryonic callus proliferation medium is a liquid medium; Optionally, carbon nanotubes are added to the embryonic callus proliferation medium; Optionally, the final concentration of the carbon nanotubes in the embryonic callus proliferation medium is 10 to 40 mg / L; Optionally, the embryonic callus proliferation medium includes the following components and their final concentrations: MS medium, 0.1-3 mg / L 2,4-D, 20-40 g / L sucrose, 0.1-1 g / L hydrolyzed casein and 10-40 mg / L carbon nanotubes; Optionally, the culture conditions in the embryonic callus proliferation medium include: a culture temperature of 20-30°C, 140-180 r / min, a light exposure time of 12-20 h, and a light intensity of 80-160 μmol m -2 s -1 .

3. The induction method according to claim 1, characterized in that: The callus induction medium is supplemented with hydrolyzed casein; Optionally, the final concentration of hydrolyzed casein in the callus induction medium is 0.1-1 g / L; Optionally, the callus induction medium includes the following components and their final concentrations: MS medium, 1-5 mg / L 2,4-D, 20-40 g / L sucrose, 5-8 g / L agar, and 0.1-1 g / L hydrolyzed casein; Optionally, the culturing step in the embryonic callus induction medium includes: culturing in the dark for 1 to 5 days, then culturing under light, and the culturing conditions under light include: a culturing temperature of 20 to 30° C., a lighting time of 12 to 20 h, and a light intensity of 80 to 160 μmol m -2 s -1 .

4. The induction method according to any one of claims 1 to 3, characterized in that: The outer diameter of the Schisandra chinensis explant is 1 to 10 mm; Optionally, the good traits include embryonic callus with milky white color and / or obvious granular structure.

5. A method for regenerating Schisandra chinensis plants, characterized in that: It includes: The embryonic callus is obtained by the induction method according to any one of claims 1 to 4.

6. The regeneration method according to claim 5, characterized in that: The regeneration method further comprises: transferring the embryonic callus to a somatic embryo induction medium for somatic embryo induction culture to obtain somatic embryos; Optionally, the somatic embryo induction medium is a solid medium; Optionally, the somatic embryo induction medium is supplemented with any one or more of sucrose, agar and exogenous nutrients, and the exogenous nutrients include any one or more of glutamine, hydrolyzed casein and inositol; Optionally, the final concentration of sucrose in the somatic embryo induction medium is 10 to 30 g / L; Optionally, the final concentration of agar in the somatic embryo induction medium is 5 to 8 g / L; Optionally, the final concentration of the exogenous nutrients in the somatic embryo induction medium is 500-1000 mg / L; Optionally, the culture conditions in the somatic embryo induction medium include: 20-30° C., dark culture for 50-70 days to obtain somatic embryos.

7. The regeneration method according to claim 6, characterized in that: The regeneration method further comprises: transferring the somatic embryo to a light condition for culture, wherein the light intensity is 100 to 140 μmol m -2 s -1 , until it differentiates into regenerated plants; Optionally, the regeneration method further comprises transplanting the regenerated plants.

8. A method for genetic transformation of Schisandra chinensis, characterized in that: It includes: The embryonic callus obtained by the induction method according to any one of claims 1 to 4 is used as an explant for genetic transformation.

9. The genetic transformation method according to claim 8, characterized in that The genetic transformation comprises the following steps: infecting the explant with an Agrobacterium infection solution containing the target gene; Optionally, the infection time is 15 to 30 minutes; Optionally, the OD of the infection fluid 600 The value is 0.4~0.8; Optionally, before the infection, the genetic transformation method further comprises: pre-culturing the explants before infection; Optionally, the pre-culture medium used in the pre-culture includes the following components and their final concentrations: MS medium, 20-40 g / L sucrose, 0.1-3 mg / L 2,4-D and 20-40 mg / L carbon nanotubes; Optionally, the pre-culture conditions include: 20-30°C, 140-180 r / min, and a light intensity of 100-140 μmol / m -2 s -1 , 1 to 5 days; Optionally, the infected explants are transferred to a co-cultivation medium for co-cultivation; Optionally, acetosyringone is added to the co-cultivation medium; Optionally, the final concentration of acetosyringone in the co-culture medium is 50 to 300 μmol / L; Optionally, the co-culture medium includes the following components and their final concentrations: MS medium, 20-40 g / L sucrose, 0.1-3 mg / L 2,4-D, 0.1-10 mg / L carbon nanotubes, and 50-300 μmol / L acetosyringone; Optionally, the co-cultivation period is: 1 to 4 days; Optionally, after the co-cultivation, the genetic transformation method further comprises: performing any one or more steps of eluting, screening and differentiating the product after the co-cultivation.

10. The genetic transformation method according to claim 9, characterized in that The elution comprises placing the co-cultured product in an elution solution for elution; the elution solution comprises an elution medium and / or water; Optionally, the elution medium includes components and their final concentrations are as follows: 1 / 3M culture medium, 10-30 g / L sucrose, 0.5-0.9 g / L inositol and 0.1-0.5 g / L timentin; Optionally, the number of elutions using the elution medium and the water is independently 1 to 4 times; Optionally, the screening adopts a screening medium for screening culture to screen out resistant tissues, and the screening medium includes components and their final concentrations are: 1 / 3M culture medium, 20-40 g / L sucrose, 5-8 g / L agar, 0.5-1 g / L glutamine, 0.1-0.5 g / L kanamycin and 0.1-0.5 g / L timentin; Optionally, the screening culture conditions include: 20-30°C, dark culture, 12-16 days; Optionally, the differentiation comprises: placing the screened product in a first differentiation medium for differentiation culture, and then transferring it to a second differentiation medium for differentiation culture until it differentiates into a plant; The first differentiation medium comprises: 1 / 3M medium, 20-40 g / L sucrose, 5-8 g / L agar, and 0.5-1 g / L glutamine; The second differentiation medium includes components and the final concentrations of the components are as follows: MS medium, 20-40 g / L sucrose and 5-8 g / L agar; Optionally, the culture conditions when using the first differentiation medium include: 20-30° C., dark culture, 50-70 days; Optionally, the culture conditions when using the second differentiation medium include: 20-30° C., and culture under light.

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