CN102250826B - Process for culturing gentiana macrophylla suspension cells - Google Patents
Process for culturing gentiana macrophylla suspension cells Download PDFInfo
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- CN102250826B CN102250826B CN 201110188467 CN201110188467A CN102250826B CN 102250826 B CN102250826 B CN 102250826B CN 201110188467 CN201110188467 CN 201110188467 CN 201110188467 A CN201110188467 A CN 201110188467A CN 102250826 B CN102250826 B CN 102250826B
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Abstract
The invention discloses a process for culturing gentiana macrophylla suspension cells. The process comprises the following steps: inducing gentiana macrophylla laminae to obtain callus, and performing multiplication culture on the callus; subculturing to obtain a loose callus cell line suitable for suspension culture; culturing suspension cells of the gentiana macrophylla after cells in a logarithmic growth phase are obtained; and extracting and producing secondary metabolites after the cells are obtained. By utilizing the process for culturing the gentiana macrophylla suspension cells provided by the invention, the cells have high growing speed and good dispersibility, the biomass of the obtained suspension cells is 15.23g/L.month (stem cell), and the content of gentiopicrin is up to 0.034%. After an elicitor is added into a culture solution, the yield of the secondary metabolites can be increased, wherein after an aspergillus niger elicitor is added, the yield of the gentiopicrin is increased by 23.99% compared with a control group in which no elicitor is added.
Description
Technical field
The invention belongs to the plant cell culture technology field, relate to a kind of culture process of bark of ash suspension cell.
Background technology
Bark of ash is the dry root of gentianaceae plant bark of ash (Gentiana macropHykgkga Pakgkg), gentiana straminea maxim (Gentiana straminea Maxim.), gentiana crassicaulis Duthie (Gentiana crassicaukgis Duthie ex Burk.) or radix gentiane dahuvicae (Gentiana dahurica Fisch.), main product in Shaanxi, the ground such as Gansu, Sichuan, the Inner Mongol.The main effective constituent gentiopicrin of bark of ash, gentiopicrin have analgesia, anti-inflammatory, hepatic cholagogic, the effect such as antibiotic, antiviral, anti-oxidant, utilize the gentiopicrin research and development two kind new medicines---Macrophylla dragon capsule is put on market.
The gentiopicrin multi-source owing to used medicinal material growth cycle long (3～5 years), and is grown in the high altitude localities in the wild of bark of ash and rough gentian or cultivation medicinal material at present, and the problem of gentiana macrophylla medicine resource has limited the development and application of effective component gentiopicrin.Producing gentiopicrin by cell culture processes, is the effective way that solves the gentiopicrin source.
Summary of the invention
The problem that the present invention solves is to provide a kind of culture process of bark of ash suspension cell, and the bark of ash suspension cell growth speed of this cultural method is fast, good dispersity, and its secondary metabolites content is high, and especially gentiopicroside in different morphological is high.
The present invention is achieved through the following technical solutions:
A kind of culture process of bark of ash suspension cell may further comprise the steps:
1) aseptic bark of ash blade is cut into 0.5～1.0cm
2The fragment of size, then carry out inducing of callus: the fragment of bark of ash leaf is inoculated in comprises 2 of 1.5～3.0mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.3～0.6mg/kg, at 25～28 ℃ of lower every day of continuous illumination 10～16h, induce 6～8d;
2) after inducing callus, callus is carried out multiplication culture: callus is inoculated in comprises 2 of 0.05～0.2mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.1～1.0mg/kg, at 22～28 ℃ of lower dark culturing 25～32d;
3) after the callus multiplication culture, comprising 2 of 0.05～0.5mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.1～1.0mg/kg, succeeding transfer culture under 22～28 ℃, dark condition, every culture time is 25～32d, and succeeding transfer culture obtains loose callus after 5～8 generations;
4) according to the ratio of every 100ml nutrient solution inoculation 4.0～6.0g, the cell of loose callus is inoculated in comprises 2 of 0.5～2.0mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D and 0.1～1.0mg/kg, suspension culture 12～18d on 22～28 ℃, dark condition, shaking table collects and obtains kind of a cell;
5) according to the ratio of every 100ml nutrient solution inoculation 4.0～6.0g, it is 6.5～7.5 that the kind cell that will be in logarithmic phase is inoculated in pH, comprise 2 of 0.5～2.0mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D, 0.1～1.0mg/kg and the sucrose of 35～50g/kg, harvested cell after suspension culture 25～30d on 22～28 ℃, dark condition, shaking table.
The acquisition of described aseptic bark of ash blade is:
Get that the bark of ash blade cleans its surface with washing powder and with flowing water flushing, volumetric concentration 75% ethanol disinfection 10s, aseptic water washing is used the mercuric chloride solution soaking disinfection 6min of mass concentration 0.1% again, uses aseptic water washing again.
During the inducing of described callus, intensity of illumination is 2000～5000Lx, and relative humidity is 65～80%.
Described suspension culture is: the kind cell that will be in logarithmic phase, to be inoculated in pH be 6.5～7.5, comprise 2 of 0.5～2.0mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D, 0.1～1.0mg/kg and the sucrose of 35～50g/kg, bottling is cultivated, liquid amount is 1/2～1/5, is suspension culture on the shaking table of 90～120r/min at 22～28 ℃, dark condition, rotating speed; Adding final concentration after cultivating at least 18d is the aspergillus niger elicitor of 60～100mg/L, continues to cultivate harvested cell behind 8～10d.
Being prepared as of described aspergillus niger elicitor:
Gather in the crops after aspergillus niger is connected to the lower dark 4～6d of cultivation of 100～120r/min on the mid-shaking table of PDA nutrient solution, 22～28 ℃, suction filtration is collected thalline, adds the pure water of 9～12 times of volumes, and suction filtration after the sterilization obtains the aspergillus niger elicitor after the filtrate sterilization.
Compared with prior art, the present invention has following useful technique effect:
The culture process of bark of ash suspension cell provided by the invention; after harvested cell, extract again and produce secondary metabolite; so not only can save a large amount of land resources; preserve the ecological environment; but manual shift control cell and gentiopicrin production process are for the problem that solves the gentiopicrin source provides and an effective way.
The culture process of bark of ash suspension cell provided by the invention, high cell growth speed, good dispersity, the biomass of gained suspension cell reach the 15.23g/L. month (stem cell), and gentiopicroside in different morphological reaches 0.034%.
Further, in nutrient solution, add after the elicitor, can improve the output of secondary metabolite: after adding the aspergillus niger elicitor, compare with the control group that does not add elicitor, the output increased of gentiopicrin 23.99%.
Description of drawings
Fig. 1 be induce and breed after the synoptic diagram of bark of ash callus;
Fig. 2 is the synoptic diagram of bark of ash kind cell suspension culture;
Fig. 3 is the micro-enlarged diagram in the bark of ash kind cell suspension culture process.
Embodiment
The invention discloses a kind of culture process of bark of ash suspension cell, getting the bark of ash blade induces to such an extent that it is carried out multiplication culture behind the callus, succeeding transfer culture obtains the callus cell system of loose suitable suspension culture, after obtaining logarithmic phase kind cell, carry out the bark of ash suspension cell culture, after harvested cell, extract again and produce secondary metabolite.The present invention is described in further detail below in conjunction with specific embodiment, and the explanation of the invention is not limited.
Embodiment 1
The culture process of bark of ash suspension cell may further comprise the steps:
1) the used bark of ash of the present invention picks up from the cultivation bark of ash in 2 years of bark of ash planting base, Feng County, Baoji growth.The time of taking is April, is sugarcane explants through callus induction with the bark of ash true leaf.
Get that the bark of ash blade cleans its surface with washing powder and with flowing water flushing, volumetric concentration 75% ethanol disinfection 10s, aseptic water washing is used the mercuric chloride solution soaking disinfection 6min of mass concentration 0.1% again, uses aseptic water washing again, obtains aseptic bark of ash blade.
Aseptic bark of ash blade is cut into 0.5～1.0cm
2The fragment of size, then carry out inducing of callus: the fragment of bark of ash leaf is inoculated in comprises 2 of 1.5mg/kg, 4-D (2, the 4-dichlorphenoxyacetic acid) and on the MS solid medium of the 6-BA of 0.3mg/kg (6-benzyl aminopurine), at 25～28 ℃ of lower every day of continuous illumination 12h, relative humidity 65% is induced 6d under intensity of illumination 3000 conditions;
2) after inducing callus, callus is carried out multiplication culture: callus is inoculated in comprises 2 of 0.05mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.1mg/kg, at 22～25 ℃ of lower dark culturing 25～28d;
3) after the callus multiplication culture, comprising 2 of 0.05mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.1mg/kg, succeeding transfer culture under 22～25 ℃, dark condition, every culture time is 25～28d, and succeeding transfer culture obtains loose callus after 7 generations;
4) according to the ratio of every 100ml nutrient solution inoculation 4.0g, the cell of loose callus is inoculated in comprises 2 of 0.5mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D and 0.1mg/kg, suspension culture 12～18d on 22～28 ℃, dark condition, shaking table collects and obtains kind of a cell;
5) according to the ratio of every 100ml nutrient solution inoculation 4.0g, it is 6.5 that the kind cell that will be in logarithmic phase is inoculated in pH, comprise 2 of 0.5mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D, 0.1mg/kg and the sucrose of 35g/kg, harvested cell after suspension culture 18～25d on 22～28 ℃, dark condition, shaking table.
Embodiment 2
The culture process of bark of ash suspension cell may further comprise the steps:
1) the used bark of ash of the present invention picks up from the cultivation bark of ash in 2 years of bark of ash planting base, Feng County, Baoji growth.The time of taking is April, is sugarcane explants through callus induction with the bark of ash true leaf.
Get that the bark of ash blade cleans its surface with washing powder and with flowing water flushing, volumetric concentration 75% ethanol disinfection 10s, aseptic water washing is used the mercuric chloride solution soaking disinfection 6min of mass concentration 0.1% again, uses aseptic water washing again.
Aseptic bark of ash blade is cut into 0.5～1.0cm
2The fragment of size, then carry out inducing of callus: the fragment of bark of ash leaf is inoculated in comprises 2 of 3.0mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.6mg/kg, at 25～28 ℃ of lower every day of continuous illumination 12h, relative humidity 65～80% is induced 8d under intensity of illumination 2000～5000Lx condition;
2) after inducing callus, callus is carried out multiplication culture: callus is inoculated in comprises 2 of 0.2mg/kg, on the MS solid medium of the 6-BA of 4-D and 1.0mg/kg, at 25～28 ℃ of lower dark culturing 28～32d;
3) after the callus multiplication culture, comprising 2 of 0.5mg/kg, on the MS solid medium of the 6-BA of 4-D and 1.0mg/kg, succeeding transfer culture under 25～28 ℃, dark condition, every culture time is 28～32d, and succeeding transfer culture obtains loose callus after 8 generations;
4) according to the ratio of every 100ml nutrient solution inoculation 6.0g, the cell of loose callus is inoculated in comprises 2 of 2.0mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D and 1.0mg/kg, suspension culture 16～18d on 25～28 ℃, dark condition, shaking table collects and obtains kind of a cell;
5) will be in the kind cell of logarithmic phase, to be inoculated in pH be 6.8～7.0, comprise 2 of 2.0mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D, 1.0mg/kg and the sucrose of 50g/kg, bottling is cultivated, liquid amount is 1/2, is suspension culture on the shaking table of 100～120r/min at 25～28 ℃, dark condition, rotating speed; Adding final concentration after cultivating 18d is the aspergillus niger elicitor of 60～80mg/L, harvested cell behind continuation cultivation 8～10d.
Being prepared as of described aspergillus niger elicitor:
Aspergillus niger (without the requirement of specific kind) is connected to 100r/min on the mid-shaking table of PDA nutrient solution, secretly gathers in the crops behind cultivation 4～6d for 22～25 ℃ times, suction filtration is collected thalline, the pure water that adds 9～10 times of volumes, suction filtration after the sterilization obtains the aspergillus niger elicitor after the filtrate sterilization.
Embodiment 3
The culture process of bark of ash suspension cell may further comprise the steps:
1) aseptic bark of ash blade is cut into 0.5～1.0cm
2The fragment of size, then carry out inducing of callus: the fragment of bark of ash leaf is inoculated in comprises 2 of 2.0mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.5mg/kg, at 26～28 ℃ of lower every day of continuous illumination 12h, relative humidity 65% is induced 7d under the intensity of illumination 3000Lx condition;
2) after inducing callus, callus is carried out multiplication culture: callus is inoculated in comprises 2 of 0.1mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.5mg/kg, at 22～24 ℃ of lower dark culturing 28～30d;
3) after the callus multiplication culture, comprising 2 of 0.2mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.6mg/kg, succeeding transfer culture under 22～24 ℃, dark condition, every culture time is 28～30d, and succeeding transfer culture obtains loose callus after 6 generations;
4) according to the ratio of every 100ml nutrient solution inoculation 5.0g, the cell of loose callus is inoculated in comprises 2 of 1.0mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D and 0.6mg/kg, suspension culture 15～16d on 22～25 ℃, dark condition, shaking table collects and obtains kind of a cell;
5) will be in the kind cell of logarithmic phase, to be inoculated in pH be 7.0～7.2, comprise 2 of 1.0mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D, 0.6mg/kg and the sucrose of 40g/kg, bottling is cultivated, liquid amount is 1/3, is suspension culture on the shaking table of 90～100r/min at 25～28 ℃, dark condition, rotating speed; Adding final concentration after cultivating 20d is the aspergillus niger elicitor of 80～100mg/L, harvested cell behind continuation cultivation 8～9d.
Being prepared as of described aspergillus niger elicitor:
Gather in the crops after aspergillus niger is connected to the lower dark 4～6d of cultivation of 100r/min on the mid-shaking table of PDA nutrient solution, 22～25 ℃, suction filtration is collected thalline, adds the pure water of 9～10 times of volumes, and suction filtration after the sterilization obtains the aspergillus niger elicitor after the filtrate sterilization.
Embodiment 4
The culture process of bark of ash suspension cell may further comprise the steps:
1) aseptic bark of ash blade is cut into 0.5～1.0cm
2The fragment of size, then carry out inducing of callus: the fragment of bark of ash leaf is inoculated in comprises 2 of 1.8mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.45mg/kg, at 25～26 ℃ of lower every day of continuous illumination 12～14h, relative humidity 65% is induced 6d under the intensity of illumination 2000Lx condition;
2) after inducing callus, callus is carried out multiplication culture: callus is inoculated in comprises 2 of 0.12mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.2mg/kg, at 24～25 ℃ of lower dark culturing 25～26d;
3) after the callus multiplication culture, comprising 2 of 0.12mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.3mg/kg, succeeding transfer culture under 22～24 ℃, dark condition, every culture time is 28～30d, and succeeding transfer culture obtains loose callus after 5 generations;
4) according to the ratio of every 100ml nutrient solution inoculation 5.5g, the cell of loose callus is inoculated in comprises 2 of 1.2mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D and 0.4mg/kg, suspension culture 16～18d on 22～25 ℃, dark condition, shaking table collects and obtains kind of a cell;
5) will be in the kind cell of logarithmic phase, to be inoculated in pH be 7.2～7.5, comprise 2 of 1.5mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D, 0.6mg/kg and the sucrose of 40g/kg, bottling is cultivated, liquid amount is 1/4, is suspension culture on the shaking table of 90～100r/min at 25～28 ℃, dark condition, rotating speed; Adding final concentration after cultivating 25d is the aspergillus niger elicitor of 80～90mg/L, harvested cell behind continuation cultivation 9～10d.
Being prepared as of described aspergillus niger elicitor:
Gather in the crops after aspergillus niger is connected to the lower dark 4～6d of cultivation of 100r/min on the mid-shaking table of PDA nutrient solution, 22～25 ℃, suction filtration is collected thalline, adds the pure water of 9～10 times of volumes, and suction filtration after the sterilization obtains the aspergillus niger elicitor after the filtrate sterilization.
Claims (3)
1. the culture process of a bark of ash suspension cell is characterized in that, may further comprise the steps:
1) aseptic bark of ash blade is cut into 0.5～1.0cm
2The fragment of size, then carry out inducing of callus: the fragment of bark of ash leaf is inoculated in comprises 2 of 1.5～3.0mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.3～0.6mg/kg, at 25～28 ℃ of lower every day of continuous illumination 10～16h, induce 6～8d;
2) after inducing callus, callus is carried out multiplication culture: callus is inoculated in comprises 2 of 0.05～0.2mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.1～1.0mg/kg, at 22～28 ℃ of lower dark culturing 25～32d;
3) after the callus multiplication culture, comprising 2 of 0.05～0.5mg/kg, on the MS solid medium of the 6-BA of 4-D and 0.1～1.0mg/kg, succeeding transfer culture under 22～28 ℃, dark condition, every culture time is 25～32d, and succeeding transfer culture obtains loose callus after 5～8 generations;
4) according to the ratio of every 100ml nutrient solution inoculation 4.0～6.0g, the cell of loose callus is inoculated in comprises 2 of 0.5～2.0mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D and 0.1～1.0mg/kg, suspension culture 12～18d on 22～28 ℃, dark condition, shaking table collects and obtains kind of a cell;
5) according to the ratio of every 100ml nutrient solution inoculation 4.0～6.0g, to be in the kind cell of logarithmic phase, to be inoculated in pH be 6.5～7.5, comprise 2 of 0.5～2.0mg/kg, in the MS liquid nutrient medium of the 6-BA of 4-D, 0.1～1.0mg/kg and the sucrose of 35～50g/kg, bottling is cultivated, liquid amount is 1/2～1/5, is suspension culture on the shaking table of 90～120r/min at 22～28 ℃, dark condition, rotating speed; Adding final concentration after cultivating at least 18d is the aspergillus niger elicitor of 60～100mg/L, continues to cultivate harvested cell behind 8～10d;
Being prepared as of described aspergillus niger elicitor:
Gather in the crops after aspergillus niger is connected to the lower dark 4～6d of cultivation of 100～120r/min on the mid-shaking table of PDA nutrient solution, 22～28 ℃, suction filtration is collected thalline, adds the pure water of 9～12 times of volumes, and suction filtration after the sterilization obtains the aspergillus niger elicitor after the filtrate sterilization.
2. the culture process of bark of ash suspension cell as claimed in claim 1 is characterized in that, the acquisition of described aseptic bark of ash blade is:
Get that the bark of ash blade cleans its surface with washing powder and with flowing water flushing, volumetric concentration 75% ethanol disinfection 10s, aseptic water washing is used the mercuric chloride solution soaking disinfection 6min of mass concentration 0.1% again, uses aseptic water washing again.
3. the culture process of bark of ash suspension cell as claimed in claim 1 is characterized in that, during the inducing of described callus, intensity of illumination is 2000～5000Lx, and relative humidity is 65～80%.
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| CN115612708B (en) * | 2022-12-19 | 2023-06-27 | 云南省农业科学院药用植物研究所 | Method for producing gentiopicroside by utilizing gentiana rigescens calli |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005012507A1 (en) * | 2003-07-25 | 2005-02-10 | The University Of Melbourne | Production of plant secondary metabolites using adsorption and elicitation in cell suspension culture |
| CN101130763A (en) * | 2007-08-08 | 2008-02-27 | 南京林业大学 | Rapid breeding method for gingkgo callus or suspending cell |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005012507A1 (en) * | 2003-07-25 | 2005-02-10 | The University Of Melbourne | Production of plant secondary metabolites using adsorption and elicitation in cell suspension culture |
| CN101130763A (en) * | 2007-08-08 | 2008-02-27 | 南京林业大学 | Rapid breeding method for gingkgo callus or suspending cell |
Non-Patent Citations (3)
| Title |
|---|
| 齐香君 等.秦艽愈伤组织诱导及增殖研究.《陕西科技大学学报》.2008,第26卷(第1期), * |
| 齐香君 等.秦艽细胞悬浮培养研究(Ⅰ).《中草药》.2010,第41卷(第3期), * |
| 齐香君 等.秦艽细胞悬浮培养研究(Ⅱ).《中草药》.2010,第41卷(第4期), * |
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