CN103461122A - Rapid propagation method of dangshen callus and suspension cells - Google Patents
Rapid propagation method of dangshen callus and suspension cells Download PDFInfo
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- CN103461122A CN103461122A CN 201310410879 CN201310410879A CN103461122A CN 103461122 A CN103461122 A CN 103461122A CN 201310410879 CN201310410879 CN 201310410879 CN 201310410879 A CN201310410879 A CN 201310410879A CN 103461122 A CN103461122 A CN 103461122A
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Abstract
The invention discloses a rapid propagation method of dangshen callus and suspension cells. The method comprises the steps of obtaining an asepsis explant; inducing calluses; culturing suspension cells; regulating and controlling the activity of enzymes and the content of dangshen polysaccharides in the suspension cells with salicylic acid. High content of the dangshen polysaccharides in the dangshen suspension cells prepared by using the method is achieved. The dangshen suspension cells are purposely regulated and controlled by utilizing the salicylic acid, so that a large number of raw materials for preparing medicines can be obtained in short time, and the production cost can be effectively lowered.
Description
Technical field
The present invention relates to the preparation method of Radix Codonopsis suspension cell, the especially regulation and control of salicylic acid to polyoses content in the Radix Codonopsis suspension cell.
Background technology
Radix Codonopsis (Codonopsis pilosula) is called three leaf vegetables, leaf list, smelly Radix Codonopsis, is the dry root of Campanulaceae Radix Codonopsis, element flower Radix Codonopsis or radix codonpsis tangshen.Its nature and flavor sweet flat, nontoxic, tonifying middle-Jiao and Qi is arranged, the effect such as promote the production of body fluid to quench thirst.With traditional Chinese medicine materials such as ginseng, the reds sage root, compare, Radix Codonopsis is traditional herbal drug of the less people's research of modern medicine.What in Radix Codonopsis, content was maximum is Codonopsis pilosula polysaccharide, Codonopsis pilosula polysaccharide is also one of active ingredient in Radix Codonopsis, research in recent years shows, Codonopsis pilosula polysaccharide has hypoglycemic, effect for reducing blood fat, the effects such as antiulcer activity, it has the effects such as antiviral, anti-ageing, anti-oxidant, antitumor in regulating immunologic function, and the present invention provides basis for its exploitation of medicinal ingredient from now on.
Salicylic acid (SA) is many physiology courses in a kind of aldehydes matter involved in plant body extensively be present in plant corpus, and particularly as On Plant Systemica Cquired Resistance, required signaling molecule causes people's extensive concern.In plant, add salicylic acid can change the activity of enzyme, improve the resistance of plant, disease resistance, can also promote the synthetic of the interior secondary metabolites of plant corpus.
Summary of the invention
Technical problem to be solved by this invention is to provide the method for quickly breeding of Radix Codonopsis cell suspension cultures, and by salicylic acid, suspension cell regulation and control is obtained to well-grown, the suspension cell that polyoses content is high, can scale provide the source of polysaccharide for pharmaceutical factory.
For solving the problems of the technologies described above, the present invention adopts following technical proposal:
Get Radix Codonopsis blade and bud, detergent immersion 4 minutes, flowing water rinses 1 hour, through alcohol 20s, mercury chloride sterilization 10min, aseptic water washing 5 times, inoculate in the MS medium and carry out callus induction, additional hormone 0.5mg/LNAA, 3mg/LIBA, sucrose 30g/L, agar 6.5g/L, pH=5.8-6.0,23 ℃ of temperature, humidity 40%～60%, luminous intensity 1000LX; The dark phase 12h of photophases 12 h; Get the light green loose callus derived, be inoculated into and fill in 50 mLMS liquid nutrient medium 100 mL triangular flasks, culture medium prescription is B
5+ 1mg/L IAA+1mg/LKT, shaking speed is 120r/min, temperature is 25 ℃, dark culturing, every the 7d subculture once, continuous culture 4-5 forms stable cell-line after generation gradually, suspends and cultivates inducing of system.Add the suspension induced to cultivate in system the 2mg/L salicylic acid solution, within 25 days, sample and weigh afterwards, measure the activity of enzyme and the content of polysaccharide.
Described medium disinfection way is at 121 ℃ of lower sterilizing 20min.
Described rare earth is the cerous nitrate rare earth preferably.
Described extraction method of polysaccharides is that precision takes dry-eye disease 1g, with clean mortar, carefully grind, proceed in 200 mL volumetric flasks, add 50 mL methyl alcohol (100%), vibration 45 min on the ultrasonic liquid oscillator, temperature 60 C, isolated by filtration, collect filtrate, finally wash down with 100% methyl alcohol carefulness, merge each washing lotion in the brown volumetric flask of 25 mL as liquid to be detected.
Described polysaccharide determination method is the anthrone sulfuric acid process, uses glucose preparation variable concentrations production standard curve.Get the extract 1.0mL extracted and add anthrone reagent 5mL, chromogenic assay absorbance under the 620nm wavelength, repeat 3 times, averages.
Result is calculated
Polyoses content (%)=CV
t/ 10
6wV
1
In formula: C: from calibration curve, check in glucose amount (μ g);
V
t: sample extracting solution cumulative volume (mL);
V
1: sampling amount during colour developing (mL);
W: sample heavy (g).
The assay method of described enzymic activity adopts spectrophotometry
The Radix Codonopsis suspension cell that adopts the present invention to prepare, fast growth, polyoses content is high, is beneficial to large production operation, and energy consumption is little, pollutes littlely, and Radix Codonopsis suspension cell increment is 4 times of contrast, and polysaccharide yield is 12.87%.
Below in conjunction with embodiment, the present invention is further elaborated, but the scope of protection of present invention is not limited to following embodiment.
Embodiment
Embodiment 1
Get outdoor Radix Codonopsis and newly go out young leaflet tablet spring, tender shoots, detergent immersion 4 minutes, flowing water rinses 1 hour, through alcohol 20s, and mercury chloride sterilization 10min, aseptic water washing 5 times, inoculate in the MS medium and carry out callus induction, additional hormone 0.5mg/LNAA, 3mg/LIBA, sucrose 30g/L, agar 6.5g/L, pH=5.8-6.0,23 ℃ of temperature, humidity 40%～60%, luminous intensity 1000LX; The dark phase 12h of photophases 12 h; Get the light green loose callus derived, be inoculated into and fill in 50 mLMS liquid nutrient medium 100 mL triangular flasks, culture medium prescription is B
5+ 0.2mg/L IAA+0.5 mg/LKT, shaking speed is 120r/min, and temperature is 25 ℃, dark culturing. and every the 7d subculture once, continuous culture 4-5 forms stable cell-line after generation gradually, suspends and cultivates inducing of system.Add the suspension induced to cultivate in system the 1mg/L salicylic acid solution, within 25 days, sample and weigh afterwards, measure the activity of enzyme and the content of polysaccharide.Radix Codonopsis suspension cell increment is 1.5 times of contrast, and polysaccharide yield is 10.24%.
Embodiment 2
Get outdoor Radix Codonopsis and newly go out young leaflet tablet spring, tender shoots, detergent immersion 4 minutes, flowing water rinses 1 hour, through alcohol 20s, and mercury chloride sterilization 10min, aseptic water washing 5 times, inoculate in the MS medium and carry out callus induction, additional hormone 0.5mg/LNAA, 3mg/LIBA, sucrose 30g/L, agar 6.5g/L, pH=5.8 ~ 6.0,23 ℃ of temperature, humidity 40% ~ 60%, luminous intensity 1000lx; The dark phase 12h of photophases 12 h; Get the light green loose callus derived, be inoculated into and fill in 40 mLMS liquid nutrient medium 100 mL triangular flasks, culture medium prescription is B
5+ 0.2 mg/L IAA+1mg/LKT, shaking speed is 120r/min, and temperature is 25 ℃, dark culturing. and every the 7d subculture once, continuous culture 4-5 forms stable cell-line after generation gradually, suspends and cultivates inducing of system.Add the suspension induced to cultivate in system the 2mg/L salicylic acid solution, within 25 days, sample and weigh afterwards, measure the activity of enzyme and the content of polysaccharide, Radix Codonopsis suspension cell increment is 1.83 times of contrast, and polysaccharide yield is 10.79%.
Embodiment 3
Get outdoor Radix Codonopsis and newly go out young leaflet tablet spring, tender shoots, detergent immersion 4 minutes, flowing water rinses 1 hour, through alcohol 20s, and mercury chloride sterilization 10min, aseptic water washing 5 times, inoculate in the MS medium and carry out callus induction, additional hormone 0.5mg/LNAA, 3mg/LIBA, sucrose 30g/L, agar 6.5g/L, pH=5.8-6.0,23 ℃ of temperature, humidity 40%～60%, luminous intensity 1000LX; The dark phase 12h of photophases 12 h; Get the light green loose callus derived, be inoculated into and fill in 40 mLMS liquid nutrient medium 100 mL triangular flasks, culture medium prescription is B
5+ 0.5 mg/L IAA+0.5 mg/LKT, shaking speed is 120r/min, and temperature is 25 ℃, dark culturing. and every the 7d subculture once, continuous culture 4-5 forms stable cell-line after generation gradually, suspends and cultivates inducing of system.Add the suspension induced to cultivate in system the 2mg/L salicylic acid solution, within 25 days, sample and weigh afterwards, measure the activity of enzyme and the content of polysaccharide.Radix Codonopsis suspension cell increment is 2.47 times of contrast, and polysaccharide yield is 11.03%.
Embodiment 4
Get outdoor Radix Codonopsis and newly go out young leaflet tablet spring, tender shoots, detergent immersion 4 minutes, flowing water rinses 1 hour, through alcohol 20s, and mercury chloride sterilization 10min, aseptic water washing 5 times, inoculate in the MS medium and carry out callus induction, additional hormone 0.5mg/LNAA, 3mg/LIBA, sucrose 30g/L, agar 6.5g/L, pH=5.8-6.0,23 ℃ of temperature, humidity 40%～60%, luminous intensity 1000LX; The dark phase 12h of photophases 12 h; Get the light green loose callus derived, be inoculated into and fill in 40 mLMS liquid nutrient medium 100 mL triangular flasks, culture medium prescription is B
5+ 0.5 mg/L IAA+1 mg/LKT, shaking speed is 120r/min, and temperature is 25 ℃, dark culturing. and every the 7d subculture once, continuous culture 4-5 forms stable cell-line after generation gradually, suspends and cultivates inducing of system.Add the suspension induced to cultivate in system the 2mg/L salicylic acid solution, within 25 days, sample and weigh afterwards, measure the activity of enzyme and the content of polysaccharide.Radix Codonopsis suspension cell increment is 3.13 times of contrast, and polysaccharide yield is 10.65%.
Embodiment 5
Get outdoor Radix Codonopsis and newly go out young leaflet tablet spring, tender shoots, detergent immersion 4 minutes, flowing water rinses 1 hour, through alcohol 20s, and mercury chloride sterilization 10min, aseptic water washing 5 times, inoculate in the MS medium and carry out callus induction, additional hormone 0.5mg/LNAA, 3mg/LIBA, sucrose 30g/L, agar 6.5g/L, pH=5.8-6.0,23 ℃ of temperature, humidity 40%～60%, luminous intensity 1000lx; The dark phase 12h of photophases 12 h; Get the light green loose callus derived, be inoculated into and fill in 40 mLMS liquid nutrient medium 100 mL triangular flasks, culture medium prescription is B
5+ 1 mg/L IAA+0.5 mg/LKT, shaking speed is 120r/min, and temperature is 25 ℃, dark culturing. and every the 7d subculture once, continuous culture 4-5 forms stable cell-line after generation gradually, suspends and cultivates inducing of system.Add the suspension induced to cultivate in system the 2mg/L salicylic acid solution, within 25 days, sample and weigh afterwards, measure the activity of enzyme and the content of polysaccharide.Radix Codonopsis suspension cell increment is 3.58 times of contrast, and polysaccharide yield is 11.94%.
Embodiment 6
Get outdoor Radix Codonopsis and newly go out young leaflet tablet spring, tender shoots, detergent immersion 4 minutes, flowing water rinses 1 hour, through alcohol 20s, and mercury chloride sterilization 10min, aseptic water washing 5 times, inoculate in the MS medium and carry out callus induction, additional hormone 0.5mg/LNAA, 3mg/LIBA, sucrose 30g/L, agar 6.5g/L, pH=5.8-6.0,23 ℃ of temperature, humidity 40%～60%, luminous intensity 1000LX; The dark phase 12h of photophases 12 h; Get the light green loose callus derived, be inoculated into and fill in 40 mLMS liquid nutrient medium 100 mL triangular flasks, culture medium prescription is B
5+ 1 mg/L IAA+1mg/LKT, shaking speed is 120r/min, and temperature is 25 ℃, dark culturing. and every the 7d subculture once, continuous culture 4-5 forms stable cell-line after generation gradually, suspends and cultivates inducing of system.Add the suspension induced to cultivate in system the 1mg/L salicylic acid solution, within 25 days, sample and weigh afterwards, measure the activity of enzyme and the content of polysaccharide.Radix Codonopsis suspension cell increment is 2.98 times of contrast, and polysaccharide yield is 12.13%.
Embodiment 7
Get outdoor Radix Codonopsis and newly go out young leaflet tablet spring, tender shoots, detergent immersion 4 minutes, flowing water rinses 1 hour, through alcohol 20s, and mercury chloride sterilization 10min, aseptic water washing 5 times, inoculate in the MS medium and carry out callus induction, additional hormone 0.5mg/LNAA, 3mg/LIBA, sucrose 30g/L, agar 6.5g/L, pH=5.8-6.0,23 ℃ of temperature, humidity 40%～60%, luminous intensity 1000lx; The dark phase 12h of photophases 12 h; Get the light green loose callus derived, be inoculated into and fill in 40 mLMS liquid nutrient medium 100 mL triangular flasks, culture medium prescription is B
5+ 1 mg/L IAA+2 mg/LKT, shaking speed is 120r/min, and temperature is 25 ℃, dark culturing. and every the 7d subculture once, continuous culture 4-5 forms stable cell-line after generation gradually, suspends and cultivates inducing of system.Add the suspension induced to cultivate in system the 3mg/L salicylic acid solution, within 25 days, sample and weigh afterwards, measure the activity of enzyme and the content of polysaccharide.Radix Codonopsis suspension cell increment is 3.17 times of contrast, and polysaccharide yield is 12.24%.
Claims (8)
1. the method for quickly breeding of a Radix Codonopsis callus and suspension cell, comprise the content of polysaccharide in the inducing of acquisition, callus, suspension cell culture, salicylic acid regulation and control Radix Codonopsis suspension cell of aseptic explant, and its key step is as follows:
(1) the Radix Codonopsis explant material disinfection of according to Plant Tissue Breeding routine disinfection method, field being taked, obtain aseptic explant;
(2) aseptic Radix Codonopsis blade step (1) obtained, bud is inoculated in callus inducing medium and carries out callus induction, and the callus subculture derived is cultivated 4-5 generation, obtains light green loose callus;
(3) light green loose callus step (2) obtained is seeded in the B of additional hormone 0.2-1 mg/L IAA and 0.5-2 mg/L KT with the tweezers gripping
5in medium, shaking suspends cultivates, and forms stable cell-line;
(4) Radix Codonopsis suspension cell step (3) obtained is inoculated into B
5+ 0.5mg/LIAA+2mg/LKT+ salicylic acid 1-3mg/L, collect the Radix Codonopsis suspension cell and weigh, and then divides two parts, and the bright sample of-70 ℃ of Refrigerator stores of portion is surveyed the activity of enzyme, dries the preservation dry sample for a 60 ℃, and Codonopsis pilosula polysaccharide is extracted, and detects.
2. according to the preparation method of claim 1, it is characterized in that: Radix Codonopsis explant blade and bud aseptic described in step (1) obtain by the following method: get Radix Codonopsis blade and bud, detergent immersion 4 minutes, flowing water rinses 1 hour, process 20s through alcohol disinfecting, mercury chloride is disinfected 10 minutes, and aseptic water washing 5 times sucks the moisture on aseptic explant surface with blotting paper.
3. in the preparation method according to claim 1, it is characterized in that: described condition of culture is 23 ℃ of temperature, humidity 40%～60%, luminous intensity 1000lx; The dark phase 12h of photophases 12 h.
4. in the preparation method according to claim 1, it is characterized in that: the described Radix Codonopsis callus of step (2) is according to obtaining by the following method: the aseptic Radix Codonopsis blade and the bud that obtain are cut into to 1cm
2fritter, inoculate in the MS medium and carry out callus induction, 3 of every bottle graft kinds, additional hormone 0.5mg/LNAA, 3mg/LIBA, sucrose 30g/L, agar 6.5g/L, pH=8, just culture is 30 days, and subculture is 4-5 time continuously, and subculture cycle 20 days, obtain light green loose callus.
5. in the preparation method according to claim 1, it is characterized in that: the described Radix Codonopsis suspension cell of step (3) is according to obtaining by the following method: get the light green loose callus derived, be seeded in the B of additional hormone 0.2-1 mg/L IAA and 0.5-2 mg/L KT with the tweezers gripping
5in medium, shaking suspends cultivates, and adds liquid nutrient medium 50mL in the triangular flask of every 100mL, and shaking speed is 120r/min, temperature is 25 ℃, dark culturing, every the 7d subculture once, continuous culture 4-5 forms stable cell-line after generation gradually, and completing suspends cultivates inducing of system.
6. in the preparation method according to claim 1, it is characterized in that: the described salicylic acid of step (4) is implemented by the following method to the regulation and control of Radix Astragali suspension cell: add the suspension induced to cultivate in system the 1-3mg/l salicylic acid solution, the content of sampling and measuring Codonopsis pilosula polysaccharide after 30 days, precision takes dry-eye disease 1g, with clean mortar, carefully grind, proceed in 200 mL volumetric flasks, add 50 mL methyl alcohol (100%), vibration 45 min on the ultrasonic liquid oscillator, temperature 60 C, isolated by filtration, collect filtrate, finally with 100% methyl alcohol carefulness, wash down, merge each washing lotion in the brown volumetric flask of 25 mL.
7. in the preparation method according to claim 1, it is characterized in that: what the polysaccharide described in step (4) extracted employing is that 40% methyl alcohol extracts.
8. in the preparation method according to claim 1, it is characterized in that: what the detection of the polysaccharide described in step (4) adopted is the By Anthrone Sulphuric acid method.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106804426A (en) * | 2016-12-26 | 2017-06-09 | 中国科学院华南植物园 | Promote the box set and method of Companumoea root vitro proliferation |
| CN107801634A (en) * | 2017-11-13 | 2018-03-16 | 陈培党 | A kind of establishing techniques of Radix Codonopsis vitro Regeneration System |
| CN109957588A (en) * | 2019-04-28 | 2019-07-02 | 江苏春之雨生物科技发展有限公司 | A kind of fluid nutrient medium inducing radix pseudostellariae cell high yield Pseudostellaria Polysaccharide |
| CN112175992A (en) * | 2020-11-16 | 2021-01-05 | 山西医科大学 | Efficient genetic transformation method for codonopsis pilosula callus |
-
2013
- 2013-09-11 CN CN 201310410879 patent/CN103461122A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106804426A (en) * | 2016-12-26 | 2017-06-09 | 中国科学院华南植物园 | Promote the box set and method of Companumoea root vitro proliferation |
| CN106804426B (en) * | 2016-12-26 | 2019-07-30 | 中国科学院华南植物园 | Promote the box set and method of Companumoea root vitro proliferation |
| CN107801634A (en) * | 2017-11-13 | 2018-03-16 | 陈培党 | A kind of establishing techniques of Radix Codonopsis vitro Regeneration System |
| CN109957588A (en) * | 2019-04-28 | 2019-07-02 | 江苏春之雨生物科技发展有限公司 | A kind of fluid nutrient medium inducing radix pseudostellariae cell high yield Pseudostellaria Polysaccharide |
| CN112175992A (en) * | 2020-11-16 | 2021-01-05 | 山西医科大学 | Efficient genetic transformation method for codonopsis pilosula callus |
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