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CN-118844341-B - Method for tissue culture of anoectochilus formosanus

CN118844341BCN 118844341 BCN118844341 BCN 118844341BCN-118844341-B

Abstract

The invention discloses a method for tissue culture of anoectochilus formosanus, which comprises the steps of selecting wild anoectochilus formosanus, removing leaves and roots, washing, shearing off stem segments of anoectochilus formosanus, soaking in alcohol, washing with sterile water, soaking in sodium hypochlorite, washing with sterile water to obtain treated stem segments, inoculating the treated stem segments to an induction culture medium for culture, inoculating the obtained stem segments to a rooting culture medium for culture, and inoculating the obtained stem segments to a tissue culture seedling culture medium for culture to obtain complete root and leaf tissue culture seedlings.

Inventors

  • QU LIPING
  • HUANG ZUNXI
  • ZHANG CHENGBO
  • LI XIA
  • YUAN YONGLEI
  • CHEN ZIXIAN
  • WANG FEIFEI
  • WANG ZUDING
  • Ma Liangyun
  • ZHU XIAOCHONG

Assignees

  • 云南云科特色植物提取实验室有限公司
  • 云南贝泰妮生物科技集团股份有限公司

Dates

Publication Date
20260505
Application Date
20240826

Claims (10)

  1. 1. A method for tissue culture of anoectochilus formosanus, comprising: (1) Stem segment treatment Selecting wild anoectochilus formosanus, removing leaves and roots, washing, shearing stem segments of the anoectochilus formosanus, soaking the stem segments in alcohol, washing the stem segments with sterile water, soaking the stem segments in sodium hypochlorite, and washing the stem segments with sterile water to obtain treated stem segments; (2) Adventitious bud induction Inoculating the treated stem to an induction culture medium for culture; the induction culture medium consists of an MS basic culture medium, 0.25 mg/L-1 mg/L of 6-BA, 0.5-2 Mg/L of 2,4-D, 0.75-1.5 mg/L of NAA, 10-30 g/L of sucrose, 0.2 G/L-0.8 g/L of activated carbon powder and 1 g/L-2 g/L of plant gel; The cultivation is carried out under red light and blue light with the illumination intensity of 40 mu mol/m 2 /s～120μmol/m 2 /s and the relative intensity of (5-7) to (3-5); (3) Rooting induction Inoculating the stem segments obtained in the step (2) to a rooting culture medium for culture; The rooting culture medium consists of 1/2-2 MS basic culture medium, 0.2-0.4 mg/L6-BA, 0.05-0.15 mg/L IBA, 10-40 g/L sucrose and 1.5g/L plant gel; The cultivation is carried out under red light and blue light with the illumination intensity of 40 mu mol/m 2 /s～120μmol/m 2 /s and the relative intensity of (3-5) to (5-7); (4) Tissue culture seedling culture Inoculating the stem segments obtained in the step (3) to a tissue culture seedling culture medium for culture to obtain tissue culture seedlings with complete roots and leaves; The tissue culture seedling culture medium consists of 1/2-1 MS basic culture medium, 3.0-5.0 mg/L6-BA, 0.3-0.5 mg/L NAA, 10-30 g/L sucrose and 1.5g/L plant gel; The cultivation is carried out under red light and blue light with the illumination intensity of 40 mu mol/m 2 /s～120μmol/m 2 /s and the relative intensity of (1-7) to (0-5).
  2. 2. The method according to claim 1, wherein in step (1), the concentration of sodium hypochlorite is 0.1g/100mL, the stem is a mature stem, and the length of the stem is 0.5cm.
  3. 3. The method according to claim 1, wherein in the step (2), the induction medium consists of MS basal medium, 0.75 mg/L6-BA, 0.5 mg/L2, 4-D, 1.25mg/L NAA, 10g/L sucrose, 0.4g/L activated carbon powder and 1.5g/L plant gel, and the pH of the induction medium is 5.6-5.8.
  4. 4. The method according to claim 1, wherein in the step (2), the temperature of the cultivation is 22 ℃ to 26 ℃, the humidity is 70%, the time of illumination per day is 12 hours, and the time of cultivation is 15 to 20 days; the illumination intensity of the red light and the blue light is 120 mu mol/m 2 /s, and the relative intensity is 5:5.
  5. 5. The method according to claim 1, wherein in step (3), the rooting medium consists of 1/2 of MS basal medium, 0.3 mg/L6-BA, 0.1mg/L IBA, 30g/L sucrose and 1.5g/L plant gel, and the pH of the rooting medium is 5.6-5.8.
  6. 6. The method according to claim 1, wherein in the step (3), the culturing is performed at a temperature of 22 ℃ to 26 ℃ and a humidity of 70%, the time of illumination per day is 12 hours, and the culturing time is 15 to 20 days.
  7. 7. The method according to claim 1, wherein in the step (3), the illumination intensities of the red light and the blue light are 80 μmol/m 2 /s and the relative intensities are 3:7.
  8. 8. The method according to claim 1, wherein in the step (4), the tissue culture seedling culture medium consists of 1/2MS basal medium, 4.0 mg/L6-BA, 0.4mg/L NAA, 30g/L sucrose and 1.5g/L plant gel, and the pH of the tissue culture seedling culture medium is 5.6-5.8.
  9. 9. The method according to claim 1, wherein in the step (4), the culturing is performed at a temperature of 22 ℃ to 26 ℃ and a humidity of 70%, the time of illumination per day is 12 hours, and the culturing time is 4 to 5 months.
  10. 10. The method of claim 1, wherein in step (4), the red light and the blue light each have an illumination intensity of 120 μmol/m 2 /s and a relative intensity of 5:5.

Description

Method for tissue culture of anoectochilus formosanus Technical Field The invention relates to a traditional Chinese medicine tissue culture method, in particular to a method for tissue culture of anoectochilus formosanus. Background The anoectochilus formosanus (Anoectochiluspingbianensis) is a plant of the genus anoectochilus of the family lanaceae, is produced from the side of the Yunnan, belongs to a national secondary protection plant, is known as the king in medicine, is used as a medicine by whole herb, and is a traditional precious medicinal material in China. The conventional patent document 1 (Shen Tingming, liu Zhiyuan, etc.) discloses that Bian Jinxian blue contains alkaloid, trace elements, amino acids, saccharides, flavonoids and other components, and has the effects of reducing blood sugar, nourishing yin, reducing fire, diminishing inflammation, relieving pain, resisting aging, promoting fluid production, nourishing skin, prolonging life and the like. Document 2 (Chen Jiangwei. Research on the internal and external antioxidation effect and mechanism of an artificially planted anoectochilus formosanus ethanol extract [ C ] university of Guangzhou pharmaceutical science, institute of research, university of Shuoshan, 2018) discloses that anoectochilus formosanus seeds are tiny powder, the seed embryo is underdeveloped, the natural reproduction rate is low, the natural environment is seriously damaged, the growth is extremely slow, animals such as insects and birds like eat the anoectochilus formosanus ethanol extract, natural resources are rare, the market price is high, the artificial random digging is caused, the wild resources are about to be exhausted, and the requirements of medical clinic and other industries cannot be met. Document 3 (Zhou Rui, kong Qiong, et al. Tissue culture and rapid propagation technology of wild anoectochilus formosanus [ J ]. Yunnan agricultural science and technology, 2014, 4:7-10) discloses rapid propagation of anoectochilus formosanus mainly through protocorms and cluster buds to conduct mass propagation [3]. However, the conventional anoectochilus roxburghii tissue culture method has high culture pollution rate, serious explant browning and long propagation period, and cannot meet production requirements. Therefore, there is a need to explore and improve the conditions of tissue culture of anoectochilus formosanus. Disclosure of Invention The invention aims to provide a method for tissue culture of anoectochilus formosanus, which solves the problems that the existing tissue culture of anoectochilus formosanus has high culture pollution rate and serious browning and the propagation period is long so as not to meet the production requirement, greatly reduces possible pollution in the tissue culture process, reduces the browning rate of explants and shortens the growth period of tissue culture seedlings. In order to achieve the above object, the present invention provides a method for tissue culture of anoectochilus formosanus, comprising: (1) Stem segment treatment Selecting wild anoectochilus formosanus, removing leaves and roots, washing, shearing stem segments of the anoectochilus formosanus, soaking the stem segments in alcohol, washing the stem segments with sterile water, soaking the stem segments in sodium hypochlorite, and washing the stem segments with sterile water to obtain treated stem segments; (2) Adventitious bud induction Inoculating the treated stem to an induction culture medium for culture; The induction culture medium consists of an MS basic culture medium, 0.25-1 mg/L of 6-BA, 0.5-2 mg/L of 2,4-D, 0.75-1.5 mg/L of NAA, 10-30 g/L of sucrose, 0.2-0.8 g/L of activated carbon powder and 1-2 g/L of plant gel; The cultivation is carried out under red light and blue light with the illumination intensity of 40 mu mol/m 2/s～120μmol/m2/s and the relative intensity of (5-7) to (3-5); (3) Rooting induction Inoculating the stem segments obtained in the step (2) to a rooting culture medium for culture; The rooting culture medium consists of 1/2-2 MS basic culture medium, 0.2-0.4 mg/L6-BA, 0.05-0.15 mg/L IBA, 10-40 g/L sucrose and 1.5g/L plant gel; The cultivation is carried out under red light and blue light with the illumination intensity of 40 mu mol/m 2/s～120μmol/m2/s and the relative intensity of (3-5) to (5-7); (4) Tissue culture seedling culture Inoculating the stem segments obtained in the step (3) to a tissue culture seedling culture medium for culture to obtain tissue culture seedlings with complete roots and leaves; The tissue culture seedling culture medium consists of 1/2-1 MS basic culture medium, 3.0-5.0 mg/L6-BA, 0.3-0.5 mg/L NAA, 10-30 g/L sucrose and 1.5g/L plant gel; The cultivation is carried out under red light and blue light with the illumination intensity of 40 mu mol/m 2/s～120μmol/m2/s and the relative intensity of (1-7) to (0-5). Preferably, in the step (1), the concentration of the sodium hypochlorite is 0.1g/100mL, the stem segm