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CN-118844339-B - Method for improving arbutin and 6' -O-caffeoyl arbutin content in bilberry tissue culture seedlings and application

CN118844339BCN 118844339 BCN118844339 BCN 118844339BCN-118844339-B

Abstract

The invention relates to the technical field of tissue culture of bilberry, in particular to a method for improving the content of arbutin and 6' -O-caffeoyl arbutin in bilberry tissue culture seedlings and application thereof, wherein a rooting culture medium selected by the bilberry tissue culture seedlings is 1/4MS culture medium, the concentration of CaCl 2 ·2H 2 O is 165-220mg/L, the concentration of KNO 3 is 0-237.5mg/L or 712.5-950mg/L, the concentration of MgSO 4 ·7H 2 O is 0-46.25mg/L, the concentration of CuSO 4 ·5H 2 O is 0.0375-0.05mg/L, the concentration of MnSO 4 ·4H 2 O is 0-11.15mg/L, and the content of active compounds CA and Arb in the bilberry tissue culture seedlings can be regulated and increased by changing the concentration of mineral elements in the 1/4MS culture medium. Is an effective way for obtaining a large amount of high-yield CA and Arb camphor leaf bilberry materials.

Inventors

  • DING YONG
  • CHANG WEI
  • ZHAO PING
  • ZHAO ZHANPING
  • WANG FENG
  • YANG LIHONG

Assignees

  • 西南林业大学

Dates

Publication Date
20260505
Application Date
20240730

Claims (6)

  1. 1. A method for improving the content of arbutin and 6' -O-caffeoyl arbutin in the tissue culture seedlings of the bilberry of the camphor leaves is characterized by comprising the following steps that a rooting culture medium selected from the tissue culture seedlings of the bilberry of the camphor leaves is a 1/4MS culture medium, wherein the concentration of Ca mineral elements is adjusted to be 1.5-2 times of the standard concentration, and the concentration of corresponding CaCl 2 ·2H 2 O is particularly 165-220mg/L; Wherein the concentration of K mineral elements is regulated to be 0-0.5 times or 1.5-2 times of the standard concentration, and the concentration of KNO 3 is specifically 0-237.5mg/L or 712.5-950mg/L; Wherein the concentration of Mg mineral elements is regulated to be 0-0.5 times of the standard concentration, and the concentration of MgSO 4 ·7H 2 O is specifically 0-46.25Mg/L; Wherein the concentration of Cu mineral elements is regulated to be 1.5 to 2 times of the standard concentration, and the concentration of corresponding CuSO 4 ·5H 2 O is specifically 0.0375 to 0.05mg/L; wherein the concentration of Mn mineral elements is regulated to be 0-0.5 times of the standard concentration, and the corresponding concentration of MnSO 4 ·4H 2 O is specifically 0-11.15mg/L.
  2. 2. The method for increasing the content of arbutin and 6' -O-caffeoyl arbutin in tissue culture seedlings of bilberry of claim 1, wherein the concentration of Ca mineral elements in a 1/4MS culture medium is adjusted to be 2 times of the standard concentration, and the concentration of corresponding CaCl 2 ·2H 2 O is specifically 220mg/L; Wherein the concentration of K mineral elements is regulated to be 0 times or 1.5 times of the standard concentration, and the concentration of KNO 3 is specifically 0 or 712.5mg/L; Wherein the concentration of Mg mineral elements is regulated to be 0.5 times of the standard concentration, and the concentration of MgSO 4 ·7H 2 O is 46.25Mg/L; wherein the concentration of Cu mineral elements is adjusted to be 2 times of the standard concentration, and the concentration of corresponding CuSO 4 ·5H 2 O is specifically 0.05mg/L; Wherein the concentration of Mn mineral elements is regulated to be 0.5 times of the standard concentration, and the corresponding concentration of MnSO 4 ·4H 2 O is specifically 11.15mg/L.
  3. 3. The method for increasing the content of arbutin and 6' -O-caffeoyl arbutin in tissue culture seedlings of bilberry of claim 1, wherein the concentration of Ca mineral elements in a 1/4MS culture medium is adjusted to be 1.5 times of the standard concentration, and the concentration of corresponding CaCl 2 ·2H 2 O is specifically 165mg/L; Wherein the concentration of K mineral elements is regulated to be 0 times or 1.5 times of the standard concentration, and the concentration of KNO 3 is specifically 0mg/L or 712.5mg/L; Wherein the concentration of Mg mineral elements is regulated to be 0 or 0.5 times of the standard concentration, and the concentration of MgSO 4 ·7H 2 O is specifically 0Mg/L or 46.25Mg/L; wherein the concentration of Cu mineral elements is adjusted to be 2 times of the standard concentration, and the concentration of corresponding CuSO 4 ·5H 2 O is specifically 0.05mg/L; Wherein the concentration of Mn mineral elements is regulated to be 0 times or 0.5 times of the standard concentration, and the corresponding concentration of MnSO 4 ·4H 2 O is specifically 0mg/L or 11.15mg/L.
  4. 4. The method for increasing the content of arbutin and 6' -O-caffeoyl arbutin in tissue culture seedlings of bilberry of claim 1, wherein the 1/4MS culture medium further comprises IBA2mg/L, sucrose 15g/L, activated carbon 0.1g/L, agar powder 4.5g/L and pH 5.8-6.0.
  5. 5. The method for improving the content of arbutin and 6' -O-caffeoyl arbutin in the tissue culture seedlings of the bilberry of the camphor leaves, which is characterized in that the tissue culture seedlings of the bilberry of the camphor leaves are subjected to secondary culture by selecting a starting culture medium of WPM+6-BA 3.0mg/L and NAA 0.2mg/L, a secondary culture medium of WPM+ZT2.0 mg/L, a culture temperature of 25+/-2 ℃, an illumination intensity of 3000lx, an illumination time of 12h/d and a culture period of 70d.
  6. 6. Use of the method according to any one of claims 1-5 in the study of the growth of bilberry leaves and the accumulation of arbutin and 6' -O-caffeoyl arbutin in bilberry leaves.

Description

Method for improving arbutin and 6' -O-caffeoyl arbutin content in bilberry tissue culture seedlings and application Technical Field The invention relates to the technical field of tissue culture of bilberry with camphorleaves, in particular to a method for improving the content of arbutin and 6' -O-caffeoyl arbutin in the tissue culture seedling of bilberry with camphorleaves and application thereof. Background As a characteristic ethnic resource plant of the genus bilberry (vaccinum) of the family azalea (Ericaceae), bilberry (Vaccinium dunalianum Wight) of the family aescidae (Ericaceae), has important edible (Li Guoze et al, 2021; kong Lingpeng et al, 2019; cheng Guian et al, 2010), medicinal (Gao et al, 2022) and whitening chemical application value (Zhao et al, 2008; luo et al, 2015). 6'-O-caffeoyl arbutin (6' -O-caffeoylarbutin, CA) and arbutin (Arbutin, arb) are the 2 major secondary metabolites with higher levels in bilberry bodies (Zhao et al, 2008; cheng Guian et al, 2010; luo et al, 2015; li Na et al, 2016). Arb has pharmacological values of whitening, antioxidation and antibiosis, is widely applied to cosmetic and pharmaceutical industries (Liu Xiaoting and Wang Xinxuan, 2022; wei et al, 2007;Lutfun et al, 2022), and CA also has various effects of whitening (Xu et al, 2014), antioxidation (Zhao et al, 2021), serum uric acid reduction (Ling Lin, 2021) and the like, and plays an important role in the fields of food, pharmacy, chemical industry and the like. The research group introduces the bilberry with camphorleaves from a wild plant cultivation base (Yunnan Chuxiong Wu Dingxian) to a school test field (Yunnan Kunming Panlong area) where authors are located, so that the content of CA and Arb is obviously reduced, and further, the difference of the content of essential mineral elements in the two-place soil for plant growth is found. There are many factors affecting plant growth and compound accumulation, of which essential mineral elements are an important factor. Mineral elements are necessary to exert important physiological functions in plants, but also directly participate in metabolism, affecting accumulation of compounds. Too high a concentration of Ca 2+ can result in a narrow plant leaf blade and reduced chlorophyll content (Peng Bo, etc.), excessive K application can inhibit growth of hemp plant height, stem thickness and leaf area, reduce activity of sucrase, reduce accumulation of dry matter and sucrose (Xu Yun, etc., 2015), K deficiency can cause yellowing, browning and burning-like burning of seedling leaf tips or leaf edges, brown spots or plaques on leaves, curling of a small number of leaves, inhibition of root growth (Yang Huixian, etc., 2019; liu Fang, etc., 2018), etc. While the effect of different mineral elements on the growth of bilberry leaves and the accumulation of CA and Arb is unknown. The prior art does not have a method for improving CA and Arb in the tissue culture seedlings of the bilberry leaves, and the problem of resource shortage of the bilberry leaves in the prior art is necessarily solved. The prior Chinese patent document CN202010176530.7 discloses a method for improving the 6'O-caffeoyl arbutin content in the bilberry tissue culture seedlings, belonging to the technical field of plant tissue culture. And selecting a culture medium to perform subculture on the bilberry tissue culture seedlings, wherein the culture medium takes WPM as a basic culture medium, two different plant growth regulators ZT and NAA are added, wherein the adding concentration of ZT is 0.1-5.0 mg/L, the adding concentration of NAA is 0.01-2.0 mg/L, and the pH value is adjusted to be 5.0-6.0. The CA content in the leaves and stem segments of the tissue culture seedlings is respectively improved to 4-7 times and 2-5 times under the condition of the WPM of the control group by applying ZT and NAA two different plant growth regulators in different concentration ratios for regulation and control, and the method is an effective way for obtaining a large amount of high-content CA camphor leaf bilberry materials. The existing methods are all to increase 6'O-caffeoyl arbutin content in the tissue culture seedlings of the bilberry leaves by chemical pesticides and other modes, so that the planting method is complex and the pollution problem is easy to generate. Disclosure of Invention The invention aims to provide a method for improving the content of arbutin and 6' -O-caffeoyl arbutin in a bilberry tissue culture seedling and application thereof, and the content of active compounds CA and Arb in the bilberry tissue culture seedling can be regulated and controlled by changing the concentration (increase or decrease) of mineral elements such as potassium (K), calcium (Ca), magnesium (Mg), copper (Cu) and manganese (Mn) in a 1/4MS culture medium. Is an effective way for obtaining a large amount of high-yield CA and Arb camphor leaf bilberry materials. In order to achieve the technical purpose and