Improved culture medium and culture method suitable for zooxanthellae in-vitro culture

Abstract

The invention provides an improved culture medium and a culture method suitable for in-vitro culture of zooxanthellae. The improved culture medium is prepared from NaNO3, NaH2PO4.H2O, Vit.B1, Vit.H, Vit.B12, Na2EDTA. 2H2O, FeCl3. 6H2O, MnCl2. 4H2O, ZnSO4. 7H2O, CoCl2. 6H2O, CuSO4. 5H2O, Na2MoO4. 2H2O, H2SeO3, NiSO4. 6H2O, Na3VO4, K2CrO4, ampicillin, kanamycin, chloramphenicol, chinomycin and streptomycin. According to the method, the cell density of zooxanthellae can be remarkably increased, meanwhile, the maximum photosynthesis efficiency of zooxanthellae and the content of chlorophyll a and carotenoid in zooxanthellae are remarkably improved, the formula is simple and easy to prepare, the effect is good, and very good application prospects are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of zooxanthellae cultivation, and relates to an improved culture medium and a cultivation method suitable for in vitro cultivation of zooxanthellae. Background Art

[0002] Coral polyps need a lot of energy during their growth and development, part of which is provided by symbiotic zooxanthellae, which is an important energy source for corals. Coral polyps provide a safer living environment for zooxanthellae. In return, zooxanthellae provide photosynthetic carbon fixation products for coral polyps, and this part of the substance accounts for 90% of the energy required by coral polyps. In 1944, Kawaguti first carried out artificial in vitro culture of zooxanthellae, but it was not successful. In 1967, Ahles conducted in vitro culture research on zooxanthellae from 70 different hosts, and only a quarter of the zooxanthellae could survive in the seawater culture medium ASP-8A. In 1985, William successfully cultured several strains of zooxanthellae in vitro using f / 2 culture medium. At first, Chinese researchers only explored the in vitro culture of zooxanthellae such as sea anemones, but zooxanthellae could only survive for up to 14 days after being in vitro. Therefore, it is crucial to find culture medium conditions suitable for the growth of zooxanthellae. Summary of the invention

[0003] Based on the above, the object of the present invention is to provide an improved culture medium and culture method suitable for in vitro culture of zooxanthellae, in which the in vitro zooxanthellae have a long survival time and excellent growth state in the improved culture medium.

[0004] The technical solution adopted by the present invention to achieve the technical purpose is:

[0005] The present invention provides an improved culture medium suitable for in vitro culture of zooxanthellae, comprising: NaNO3, NaH2PO4·H2O, Vit.B1, Vit.H, Vit.B 12 , Na2EDTA·2H2O, FeCl3·6H2O, MnCl2·4H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, Na2MoO4·2H2O, H2SeO3, NiSO4·6H2O, Na3VO4, K2CrO4, ampicillin, kanamycin, chloramphenicol, spectinomycin, and streptomycin.

[0006] Preferably, in the modified culture medium, the final concentrations of ampicillin, kanamycin, chloramphenicol, spectinomycin and streptomycin are all 50 μg mL -1 .

[0007] Preferably, in the improved culture medium, the final concentration of Vit.B1 is 2.96×10 -7The final concentration of Vit.H is 2.05×10 -9 M, Vit.B 12 The final concentration was 3.69×10 -10 M.

[0008] Preferably, the final concentration of NaNO3 in the improved culture medium is 75 μg L -1 The final concentration of NaH2PO4·H2O was 5 μg L -1 .

[0009] Preferably, in the improved culture medium, the final concentration of Na2EDTA·2H2O is 4.36 μg L -1 The final concentration of FeCl3·6H2O was 3.15 μg L -1 The final concentration of MnCl2·4H2O was 178.10 μg L -1 The final concentration of ZnSO4·7H2O was 23.00 μg L -1 The final concentration of CoCl2·6H2O was 11.90 μg L -1 The final concentration of CuSO4·5H2O was 2.50 μg L -1 The final concentration of Na2MoO4·2H2O was 19.90 μg L -1 The final concentration of H2SeO3 was 1.29 μg L -1 The final concentration of NiSO4·6H2O was 2.63 μg L -1 The final concentration of Na3VO4 was 1.84 μg L -1 The final concentration of K2CrO4 was 1.94 μg L -1 .

[0010] The present invention also provides a culture method suitable for in vitro culture of zooxanthellae, using the above-mentioned improved culture medium, comprising: transferring the resuspended solution of zooxanthellae into a culture bottle containing the improved culture medium, placing the bottle in a 25°C light incubator for culture, and culturing the bottle in a light incubator with a light intensity of 60 μmol photons·m -2 ·s -1 , the light-dark cycle is 14h:10h.

[0011] Preferably, the resuspension of zooxanthellae is prepared by the following method: taking a D-series zooxanthellae liquid into a sterile centrifuge tube, centrifuging it at 3000 rpm / min and 25° C. for 5 min, taking it out, pouring out the waste culture liquid in an ultra-clean workbench, adding the modified culture medium thereto and resuspending the zooxanthellae.

[0012] Preferably, the initial concentration of zooxanthellae is 1×10 5 cells / mL.

[0013] The beneficial effects of the present invention are:

[0014] The present invention provides an improved culture medium suitable for in vitro culture of zooxanthellae. By adjusting the composition of different added antibiotics, not only can the cell density of zooxanthellae be significantly increased, but also the maximum light and efficiency (Fv / Fm) of zooxanthellae and the contents of chlorophyll a and carotenoids in zooxanthellae are significantly improved. The formula is simple and easy to prepare, the effect is good, and the application prospect is very good. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The density of zooxanthellae cultured in different media;

[0016] Figure 2 Fv / Fm of zooxanthellae cultured in different media;

[0017] Figure 3 Chlorophyll a and carotenoid contents of zooxanthellae cultured in different media;

[0018] 1 to 5 in the figure correspond to different groups in Table 2. DETAILED DESCRIPTION

[0019] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0020] Example

[0021] 1. Prepare the culture medium according to the basic culture medium formula in Table 1.

[0022] Table 1. Basic culture medium formula

[0023]

[0024] 2. On the basis of the basic culture medium, add different antibiotics according to Table 2, and the addition amount is 50μgmL -1 .

[0025] Table 2. Table of different antibiotics added

[0026] Group Amphotericin B Ampicillin Kanamycin Chloramphenicol Spectinomycin Streptomycin penicillin 1 — — — — — — — 2 — √ √ √ — √ √ 3 √ √ √ — √ √ — 4 — — √ √ √ √ √ 5 — √ √ √ √ √ —

[0027] 3. Take 30 ml of D-type zooxanthellae solution (concentration of 1×10 5 cells / mL) in a sterile centrifuge tube, centrifuge at 3000 rpm / min and 25°C for 5 min, then take out the culture medium, discard the waste culture medium in a clean bench, and inoculate the zooxanthellae into fresh modified culture medium with an initial inoculation concentration of 1×105 cells / mL, and cultured in a 25°C intelligent light incubator with a light intensity of approximately 60 μmol photons·m -2 ·s -1 , the light-dark cycle is 14h:10h.

[0028] 4. The density and Fv / Fm of zooxanthellae were measured on the 1st, 2nd, 5th, 9th and 14th days of culture, and the content of each pigment in zooxanthellae was determined on the last day.

[0029] 5. Compare the data to obtain the optimal culture medium conditions.

[0030] The results are as follows Figures 1 to 3 As shown, the culture medium with antibiotics added according to Group 5 can significantly increase the cell density of zooxanthellae, and at the same time, it also significantly improves the maximum light and efficiency (Fv / Fm) of zooxanthellae and the content of chlorophyll a and carotenoids in zooxanthellae. Therefore, the antibiotics in Group 5 were selected to be added to the basal culture medium as the improved culture medium.

[0031] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. An improved culture medium suitable for in vitro culture of zooxanthellae, comprising: NaNO3, NaH2PO4·H2O, Vit.B1, Vit.H, Vit.B 12 , Na2EDTA·2H2O, FeCl3·6H2O, MnCl2·4H2O, ZnSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, Na2MoO4·2H2O, H2SeO3, NiSO4·6H2O, Na3VO4, K2CrO4, ampicillin, kanamycin, chloramphenicol, spectinomycin, and streptomycin.

2. The improved culture medium according to claim 1, characterized in that In the modified culture medium, the final concentrations of ampicillin, kanamycin, chloramphenicol, spectinomycin and streptomycin were all 50 μg mL -1 .

3. The improved culture medium according to claim 1, characterized in that In the modified culture medium, the final concentration of Vit.B1 is 2.96×10 -7 The final concentration of Vit.H is 2.05×10 -9 M, Vit.B 12 The final concentration was 3.69×10 -10 M.

4. The improved culture medium according to claim 1, characterized in that In the modified culture medium, the final concentration of NaNO3 was 75 μg L -1 The final concentration of NaH2PO4·H2O was 5 μg L -1 .

5. The improved culture medium according to claim 1, characterized in that In the modified medium, the final concentration of Na2EDTA·2H2O was 4.36 μg L -1 The final concentration of FeCl3·6H2O was 3.15 μg L -1 The final concentration of MnCl2·4H2O was 178.10 μg L -1 The final concentration of ZnSO4·7H2O was 23.00 μg L -1 The final concentration of CoCl2·6H2O was 11.90 μg L -1 The final concentration of CuSO4·5H2O was 2.50 μg L -1 The final concentration of Na2MoO4·2H2O was 19.90 μg L -1 The final concentration of H2SeO3 was 1.29 μg L -1 The final concentration of NiSO4·6H2O was 2.63 μg L -1 The final concentration of Na3VO4 was 1.84 μg L -1 The final concentration of K2CrO4 was 1.94 μg L -1 .

6. A method for in vitro culture of zooxanthellae, using the improved culture medium according to any one of claims 1 to 5, comprising: The resuspended zooxanthellae was transferred into a culture bottle containing the modified culture medium and placed in a light incubator at 25°C with a light intensity of 60 μmol photons·m -2 ·s -1 , the light-dark cycle is 14h:10h.

7. The culture method according to claim 6, characterized in that: The resuspension of zooxanthellae is prepared by the following method: taking a D-series zooxanthellae liquid into a sterile centrifuge tube, centrifuging it at 3000 rpm / min and 25° C. for 5 minutes, then taking it out, pouring out the waste culture liquid in an ultra-clean workbench, adding the modified culture medium thereto and resuspending the zooxanthellae.

8. The culture method according to claim 6, characterized in that: The initial concentration of zooxanthellae was 1×10 5 cells / mL.

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