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CN-122012372-A - Camellia nitidissima suspension cell culture method

CN122012372ACN 122012372 ACN122012372 ACN 122012372ACN-122012372-A

Abstract

The embodiment of the specification provides a golden camellia suspension cell culture method which comprises the steps of collecting golden camellia leaves, inoculating the golden camellia leaves into a WPM liquid culture medium containing hormone for culture, inducing to form primary callus, inoculating the primary callus into the WPM liquid culture medium containing auxin and cytokinin for culture, and obtaining golden camellia suspension cells.

Inventors

  • LIU YU
  • WANG YUAN
  • JIN YINBING
  • LIU HANSHI

Assignees

  • 大连普瑞康生物技术有限公司

Dates

Publication Date
20260512
Application Date
20260413

Claims (10)

  1. 1. The golden camellia suspension cell culture method is characterized by comprising the following steps of: collecting golden camellia leaves, inoculating the golden camellia leaves into a WPM liquid culture medium containing hormone to be cultured, and inducing the golden camellia leaves to form primary callus, wherein the hormone comprises naphthalene acetic acid and 6-benzylaminopurine; inoculating the primary callus into a WPM liquid culture medium containing auxin and cytokinin for culture to obtain the camellia chrysantha suspension cells.
  2. 2. The method of claim 1, wherein the hormone-containing WPM broth further comprises an anti-browning agent, wherein the anti-browning agent is polyvinylpyrrolidone.
  3. 3. The method of claim 2, wherein prior to inoculating the golden camellia leaves to the hormone-containing WPM liquid medium, the method further comprises: spraying salicylic acid-containing solution to the golden camellia leaves, wherein the concentration of salicylic acid in the salicylic acid-containing solution is 0.05-0.2mmol/L; And (3) treating the golden camellia leaves by using a compound liquid containing mercury disinfectant and surfactant.
  4. 4. The method according to claim 1, wherein the culture conditions in the hormone-containing WPM liquid medium comprise a temperature of 25+ -2 ℃, a dark culture of 6-8 days followed by a light-dark alternate culture, wherein the light-dark alternate culture has a light cycle of 13-15 hours of light and 9-11 hours of darkness and a light intensity of 1500-2500lux.
  5. 5. The method according to claim 1, wherein in the WPM liquid medium containing auxin and cytokinin, the auxin is 2, 4-dichlorophenoxyacetic acid, the concentration of the auxin is 1-3mg/L, the cytokinin is kinetin, and the concentration of the cytokinin is 0.2-1mg/L.
  6. 6. The method according to claim 1, wherein the culture conditions in the WPM liquid medium containing auxin and cytokinin comprise shaking speed of 100-150rpm, temperature of 20-28 ℃, light period of 12-18 hours, darkness of 6-12 hours after light irradiation, and light irradiation intensity of 800-1200lux.
  7. 7. The method according to claim 1, wherein the method further comprises: And (3) further inducing accumulation of secondary metabolites of the cells by at least one treatment mode of cold treatment at 2-6 ℃ and methyl jasmonate treatment to obtain the target camellia nitidissima suspension cells.
  8. 8. The method of claim 7, wherein the 2-6 ℃ cold treatment is for 12-36 hours, the methyl jasmonate treatment is for 24-72 hours, and the concentration of methyl jasmonate in the methyl jasmonate treatment is 50-150 μΜ.
  9. 9. The golden camellia cell culture is characterized by comprising the golden camellia suspension cells or target golden camellia suspension cells prepared by the method according to any one of claims 1 to 8, wherein the content of secondary metabolites in a dried product of the golden camellia cell culture is 10% -20% of flavone and 15% -25% of polyphenol.
  10. 10. The camellia chrysantha cell culture of claim 9, wherein expression of PAL, ANR1 genes is up-regulated in the camellia chrysantha cell culture.

Description

Camellia nitidissima suspension cell culture method Technical Field The specification relates to the field of plant cell culture and bioactive substance preparation, in particular to a camellia nitidissima suspension cell culture method. Background Camellia nitidissima (school name: camellia petelotii) is evergreen shrub of Camellia genus of Theaceae. The active ingredients such as flavonoid (e.g. procyanidine B2, catechin, etc.), polyphenol, triterpenoid saponin, etc. enriched in the tissue have important application value in the fields of functional foods and medicines. The problems of scarce resources, low artificial planting efficiency and the like exist at present, and the large-scale requirement of active ingredients of golden camellia cannot be met. Although cell culture technology exists at present, the problems of explant treatment, low efficiency of callus culture, large industrialization obstacle and the like still exist, so that the standardized production requirement of high-purity flavonoid extract is difficult to meet. In view of the above, it is desirable to provide a suspension cell culture method for golden camellia, which solves the difficult problems of active ingredient production caused by scarce resources, low efficiency of the traditional culture technology and metabolic regulation and control misalignment of golden camellia, and realizes efficient, stable and environment-friendly large-scale preparation. Disclosure of Invention One or more embodiments of the specification provide a golden camellia suspension cell culture method, which comprises the steps of collecting golden camellia leaves, inoculating the golden camellia leaves into a WPM liquid culture medium containing hormone for culture, inducing the hormone to form primary callus, inoculating the primary callus into the WPM liquid culture medium containing auxin and cytokinin for culture, and obtaining golden camellia suspension cells. In some embodiments, the concentration of naphthalene acetic acid in the hormone-containing WPM liquid medium is 1-5mg/L and the concentration of 6-benzylaminopurine is 0.1-1mg/L. In some embodiments, the hormone-containing WPM liquid medium further comprises an anti-browning substance, the anti-browning substance being polyvinylpyrrolidone. In some embodiments, before inoculating the golden camellia leaves into the hormone-containing WPM liquid medium, the method further comprises spraying a salicylic acid-containing solution to the golden camellia leaves, wherein the concentration of salicylic acid in the salicylic acid-containing solution is 0.05-0.2mmol/L, and treating the golden camellia leaves with a complex solution of a mercury-containing disinfectant and a surfactant. In some embodiments, the culture conditions in the WPM liquid culture medium containing hormone comprise a temperature of 25+/-2 ℃, a dark culture period of 13-15 hours, 9-11 hours darkness after illumination and an illumination intensity of 1500-2500lux, and transferring to light-dark alternate culture after dark culture for 6-8 days. In some embodiments, the auxin is 2, 4-dichlorophenoxyacetic acid, the concentration of the auxin is 1-3mg/L, the cytokinin is kinetin, and the concentration of the cytokinin is 0.2-1mg/L in the WPM liquid medium containing the auxin and the cytokinin. In some embodiments, the culture conditions in the WPM liquid medium containing auxin and cytokinin comprise shaking speed of 100-150rpm, temperature of 20-28 ℃, illumination period of 12-18 hours, darkness of 6-12 hours after illumination, and illumination intensity of 800-1200lux. In some embodiments, the method further comprises the step of further inducing accumulation of secondary metabolites of the cells by at least one of cold treatment at 2-6 ℃ and methyl jasmonate treatment to obtain the target camellia nitidissima suspension cells. In some embodiments, the 2-6 ℃ cold treatment time is 12-36 hours, the methyl jasmonate treatment time is 24-72 hours, and the concentration of methyl jasmonate in the methyl jasmonate treatment is 50-150 μΜ. One or more embodiments of the present disclosure provide a camellia chrysantha cell culture, where the camellia chrysantha cell culture includes the camellia chrysantha suspension cell or the target camellia chrysantha suspension cell prepared by any one of the above camellia chrysantha suspension cell culture methods, and the dried product of the camellia chrysantha cell culture contains 10% -20% of flavone and 15% -25% of polyphenol. In some embodiments, the expression of PAL, ANR1 genes is up-regulated in the golden camellia cell culture. Drawings The present specification will be further elucidated by way of example embodiments, which will be described in detail by means of the accompanying drawings. These embodiments are not limiting, wherein: FIG. 1 is a schematic diagram of solid state cultured callus of Camellia nitidissima according to some embodiments of the present disclosure; FIG. 2 is a