CN102994443A - Suspension culture solution and suspension culture method for loquat cells - Google Patents
Suspension culture solution and suspension culture method for loquat cells Download PDFInfo
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- CN102994443A CN102994443A CN2012104577339A CN201210457733A CN102994443A CN 102994443 A CN102994443 A CN 102994443A CN 2012104577339 A CN2012104577339 A CN 2012104577339A CN 201210457733 A CN201210457733 A CN 201210457733A CN 102994443 A CN102994443 A CN 102994443A
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Abstract
The invention discloses suspension culture solution and a suspension culture method for loquat cells. The culture solution comprises MS basic culture medium, 0.5-6.0 mg/L of 2,4-D ((2,4-dichlorophenyloxy)acetic acid), 0.5-6.0 mg/L of NAA (naphthyl acetic acid), 0.5-6.0 mg/L of IBA (indole butyric acid), 0-4.0 mg/L of KT (kinetin), 0-4.0 mg/L of BA (benzylaminopurine) and 1-3.5% of cane sugar, and has the pH of 4.0-6.5. The culture method is used for culturing callus in the culture solution. According to the invention, a liquid culture system for loquat cells is established; and with the adoption of the optimal mode of the culture method disclosed by the invention for culture, the yield of ursolic acid in a loquat cell culture is 36.49 mg/g.DW.
Description
Technical field
The present invention relates to biological technical field, relate to particularly loquat cell suspension culture method.
Background technology
Ursolic acid is most important secondary metabolites in the Loquat Leaf, be experimental results show that by tumour hospital of Chinese Medicine academy of sciences to be expected to become low toxicity, effective new type natural cancer therapy drug, and market is very in short supply.Product is many at present carries out organic extraction from the loquat blade, and cost is high.The visible CN200710008809.9 of extracting method, 200910154645.X, 201210057030.7 etc.
Mainly concentrate in the prior art: to the foundation of the callus culture of loquat and asexual fast traditional font system; Extraction and pharmacological research thereof to the Loquat Leaf active ingredient.It is important technology route during present Chinese medicine is produced that formation for Plant Cell Suspension cultivation approach is produced secondary metabolite, but at present in loquat research, there is no to set up suspension culture system to extract the report of the triterpene compound such as ursolic acid.
Summary of the invention
The object of the present invention is to provide a kind of loquat cell suspension culture method, produce natural ursolic acid, to solve the above-mentioned problems in the prior art.
Technical scheme provided by the invention is as follows:
Technical scheme one:
A kind of nutrient solution for the loquat cell suspension culture comprises:
The MS minimum medium
2,4-D(2, the 4-dichlorophenoxyacetic acid) 0.5-6.0mg/L;
The NAA(a-naphthylacetic acid) 0.5-6.0mg/L;
The IBA(indolebutyric acid) 0.5-6.0mg/L;
The 6-BA(6-benzyladenine) 0-4.0mg/L;
KT(kinetin, kinetin) 0-4.0mg/L;
Sugarcane 1-3.5%(weight ratio);
pH 4.0-6.5。
Nutrient solution scheme best among the present invention is: MS minimum medium+0.5mg/L NAA+3% sucrose+PH 6.0
Technical scheme two:
A kind of loquat cell suspension culture method comprises the steps:
(1) the loquat callus is obtained in cultivation;
(2) provide aforesaid loquat cell suspending culture solution;
(3) callus carries out suspension culture in suspending nutrient solution;
(4) harvested cell extracts ursolic acid.
In preferred embodiment of the present invention, the callus that is used for suspension culture be faint yellow, growth conditions is good, open-textured loquat rataria is induced the callus that comes.Obtain callus and can adopt prior art.Behind the subculture 5 times, change suspension culture over to.
A kind of loquat cell suspension culture method, the cell suspension culture based formulas is: MS minimum medium+0.5mg/L NAA+3% sucrose, the pH value is 6.0; Extraneous control condition is: illumination condition is 400Lux(12h/d), the bottled liquid 100ml of triangle of employing 250ml, inoculum size is 40-60g/LFW, shaking speed is 130r/min, adopts the air-permeable envelope sealing, cultivates 18 days results.
Because there is multiple limitation in traditional Loquat Leaf ursolic acid extracting method, the invention provides nutrient solution and cultural method for the loquat cell suspension culture.The present invention is take the loquat rataria as material, carry out inducing of good callus, good callus changes liquid nutrient medium over to through succeeding transfer culture repeatedly, then the screening of carrying out condition of suspension culture detects the content of target product ursolic acid simultaneously, thereby set up loquat callus suspension culture system, for the industrialization production of extracting ursolic acid by the loquat cell suspension culture lays the foundation.Wherein, adopt best mode to cultivate, the yield of ursolic acid is 36.49mg/gDW in the loquat cell culture.Than in the Loquat Leaf (South Asia loquat) in the present big vast swallow duckweed bibliographical information the content (14.01mg/gDW) of high ursolic acid improved about 160.46%.
Description of drawings
Fig. 1 is loquat cell suspension culture growth curve and ursolic acid content figure in the embodiment of the invention.
Embodiment
Test materials: the loquat young fruit, pick up from Fujian Province Putian City fruit tree research institute.
The extracting method of loquat cell effective constituent ursolic acid is the prior art ultrasonic extraction, is specially: solvent is 95% ethanol, solid-liquid ratio 1:30(g/ml), ultrasonic extraction, 40 ℃ of extraction temperature, the extraction frequency is 25KHZ, extraction power 260W, extraction time 1 time, ultrasonic time 40min.Measure with liquid chromatography (HPLC).
The acquisition of embodiment 1 loquat callus
Adopt prior art, induce acquisition from the loquat young fruit, inducing culture is MS minimum medium+1mg/L2,4-D+0.5mg/L KT+ sucrose 2%+6.8g/L agar powder, 5.8,25 ℃ of PH, after the dark culturing, same substratum and culture condition succeeding transfer culture 5 times, change suspension culture over to.
Foundation and the cultural method of loquat cell suspension culture system
Material: choose faint yellow on the solid medium, growth conditions is good, quality is loose, the loquat rataria is induced and the embryo callus that comes.With original solid culture based formulas and culture condition (among the embodiment 1, inducing culture is MS minimum medium+1mg/L 2,4-D+0.5mg/L KT+ sucrose 2%, PH 5.8,25 ℃, dark culturing, shaking speed 110r/min) continuous subculture more than 5 generations, obtain the suspension of homogeneous, to carry out follow-up study on regulation (embodiment 2-9).The nutrient solution prescription and the culture condition that adopt in the study on regulation are: MS minimum medium+1mg/L 2, and 4-D+0.5mg/L KT+ sucrose 2%, 5.8,25 ℃ of PH, dark culturing, shaking speed 110r/min, when investigating certain factor, fixedly other factors is studied.
The main index of investigating: suspension culture fresh weight specific growth rate, ursolic acid content.
Ursolic acid is with ultrasonic extraction, and content is measured with liquid chromatography (HPLC)
Embodiment 2 hormones are on the impact of suspended culture cell growth and ursolic acid content
Table 12,4D is on the impact of suspended culture cell growth and ursolic acid content
| Process number | 2,4-D（mg/L） | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 0.5 | 162.11 | 11.68 |
| 2 | 1.0 | 147.58 | 8.37 |
| 3 | 2.0 | 148.75 | 6.84 |
| 4 | 4.0 | 111.08 | 5.34 |
| 5 | 6.0 | 92.40 | 7.83 |
The suitable loquat cell suspension culture of conclusion: 2,4-D 0.5
Table 2NAA is on the impact of suspended culture cell growth and ursolic acid content
| Process number | NAA（mg/L） | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 0.5 | 257.61 | 36.49 |
| 2 | 1.0 | 272.80 | 30.52 |
| 3 | 2.0 | 286.21 | 28.00 |
| 4 | 4.0 | 280.96 | 29.02 |
| 5 | 6.0 | 271.04 | 28.63 |
Conclusion: NAA0.5 is suitable for the loquat cell suspension culture
Table 3IBA is on the impact of suspended culture cell growth and ursolic acid content
| Process number | IBA（mg/L） | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 0.5 | 29.06 | 10.53 |
| 2 | 1.0 | 24.28 | 10.84 |
| 3 | 2.0 | 44.26 | 10.39 |
| 4 | 4.0 | 56.85 | 9.75 |
| 5 | 6.0 | 59.13 | 10.56 |
Conclusion: each scope of growth hormone IBA all is not suitable for the loquat cell suspension culture, and in the trial stretch, IBA6.0 is relatively suitable.
Table 4BA is on the impact of suspended culture cell growth and ursolic acid content
| Process number | BA（mg/L） | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 0 | -4.59 | 22.10 |
| 2 | 0.25 | -5.73 | 22.14 |
| 3 | 0.5 | 3.32 | 20.93 |
| 4 | 1.0 | 13.06 | 24.75 |
| 5 | 2.0 | 25.32 | 23.21 |
| 6 | 4.0 | 51.47 | 24.88 |
Conclusion: BA2.0 can be used for the loquat cell suspension culture
Table 5KT is on the impact of suspended culture cell growth and ursolic acid content
| Process number | KT（mg/L） | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 0 | 28.70 | 17.00 |
| 2 | 0.25 | 28.47 | 21.03 |
| 3 | 0.5 | 44.21 | 20.50 |
| 4 | 1.0 | 51.55 | 21.05 |
| 5 | 2.0 | 71.16 | 22.95 |
| 6 | 4.0 | 82.96 | 24.98 |
Conclusion: the KT4.0 loquat cell suspension culture that suits
Brief summary: the comprehensive specific growth rate of single-factor hormone regulating and controlling and ursolic acid content, be advisable with NAA0.5mg/L.
Embodiment 3PH value is on the impact of suspended culture cell growth and ursolic acid content
Behind selected minimum medium and the suitable hormone combination, different pH values is grown on suspended culture cell and the impact of ursolic acid content in the observation liquid nutrient medium.
Table 6pH value is on the impact of suspended culture cell growth and ursolic acid content
| Process number | PH | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 4 | 218.78 | 12.24 |
| 2 | 4.5 | 192.43 | 11.83 |
| 3 | 5 | 239.88 | 11.57 |
| 4 | 5.5 | 228.82 | 12.09 |
| 5 | 6 | 261.72 | 13.23 |
| 6 | 6.5 | 217.47 | 11.67 |
Conclusion: the loquat cell suspension culture all can be grown in the PH4.0-6.5 scope, but take PH6.0 as best pH value.Embodiment 4 illumination are on the impact of suspended culture cell growth and d-Bomeol content
Illumination may have larger impact to cell suspension culture, therefore the illumination condition of suspension culture system is studied.
Choose 0,400,1000,2000 and the light intensity of 4000Lux to culture 12h/d irradiation (simulating nature), to filter out favourable optimum illumination condition.
Table 7 illumination condition is on the impact of suspended culture cell growth and ursolic acid content
| Process number | Intensity of illumination (lux) | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 0 | 301.42 | 11.85 |
| 2 | 400 | 336.11 | 17.18 |
| 3 | 1000 | 265.28 | 12.14 |
| 4 | 2000 | 203.64 | 13.33 |
| 5 | 4000 | 180.31 | 14.03 |
Conclusion: the 400lux illumination condition is comparatively suitable.
Embodiment 5 inoculum sizes are on the impact of suspended culture cell growth and ursolic acid content
To the clone of any suspension culture, the density that has its growth to be fit to.In the 250ml triangular flask, every bottled 100ml nutrient solution that enters, observation different vaccination amount is on the impact of suspended culture cell growth and ursolic acid content.
Table 8 inoculum size is on the impact of suspended culture cell growth and ursolic acid content
| Process number | Inoculum size (g/100ml) | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 2 | 108.13 | 8.38 |
| 2 | 4 | 243.25 | 11.85 |
| 3 | 6 | 242.67 | 15.62 |
| 4 | 8 | 239.00 | 16.90 |
| 5 | 10 | 182.80 | 15.91 |
| 6 | 12 | 147.92 | 16.94 |
Conclusion: inoculum size 8g/L optimum loquat cell suspension culture
Embodiment 6 shaking speed are on the impact of suspended culture cell growth and ursolic acid content
Culture is placed respectively 50,70,90,110,130,150, carries out suspension culture on the rotary shaking table of 170r/min, and the observation shaking speed is on the impact of suspended culture cell growth and ursolic acid content.
Table 9 shaking speed is on the impact of suspended culture cell growth and ursolic acid content
The most suitable loquat cell suspension culture of conclusion: shaking speed 130rpm/min.
Embodiment 7 harvest times are on the impact of suspended culture cell growth and ursolic acid content
Respectively at the 18th day results after cultivating, the observation harvest time is on the impact of suspension culture callus, Growth of Cells and ursolic acid content.The results are shown in Figure 1.
As seen from Figure 1, the growth curve of culture presents the S type substantially, and 7-15d enters the growth platform phase for the growth logarithmic phase behind the 15d, in the growth cycle, and the rear rising that descends first of the content of ursolic acid, the content of ursolic acid also is in a large number accumulation behind 15d.
Embodiment 8 liquid amounts are on the impact of suspended culture cell growth and ursolic acid content
Material is cultivated with the culturing bottle of 250ml, and the ratio of new and old substratum is fixed as 1:1.
Table 10 liquid amount is on the impact of suspended culture cell growth and ursolic acid content
| Process number | Liquid amount (ml) | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 40 | 255.42 | 11.21 |
| 2 | 70 | 287.39 | 9.22 |
| 3 | 100 | 308.99 | 8.26 |
| 4 | 130 | 227.90 | 9.62 |
| 5 | 160 | 78.84 | 9.13 |
The culturing bottle of conclusion: 250ml is take the liquid amount of 100ml as good.
Embodiment 9 sucrose contents are on the impact of suspended culture cell growth and ursolic acid content
Table 11 sucrose content is on the impact of suspended culture cell growth and ursolic acid content
| Process number | Sucrose content (%) | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 1.0 | 191.61 | 6.34 |
| 2 | 1.5 | 167.51 | 6.88 |
| 3 | 2.0 | 210.92 | 6.90 |
| 4 | 2.5 | 200.80 | 6.63 |
| 5 | 3.0 | 230.89 | 7.57 |
| 6 | 3.5 | 190.41 | 7.22 |
Conclusion: sucrose content with 3% for the most suitable.
Claims (4)
1. nutrient solution that is used for the loquat cell suspension culture comprises:
The MS minimum medium;
2,4-D 0.5-6.0mg/L；
NAA 0.5-6.0mg/L；
IBA 0.5-6.0mg/L；
6-BA 0-4.0mg/L；
KT 0-4.0mg/L；
Sucrose 1-3.5%;
pH 4.0-6.5。
2. a nutrient solution that is used for the loquat cell suspension culture is characterized in that MS minimum medium+0.5mg/L NAA+3% sucrose+PH 6.0.
3. a loquat cell suspension culture method comprises the steps:
(1) the loquat callus is obtained in cultivation;
(2) provide the loquat cell suspending culture solution of claim 1 or 2;
(3) callus carries out suspension culture in suspending nutrient solution;
(4) harvested cell extracts ursolic acid.
4. loquat cell suspension culture method, the cell suspension culture based formulas is: MS minimum medium+0.5mg/L NAA+3% sucrose, the pH value is 6.0; Extraneous control condition is: illumination condition is 400Lux, 12h/d, and the bottled liquid 100ml of triangle of employing 250ml, inoculum size is 40-60g/LFW, shaking speed is 130r/min, adopts the air-permeable envelope sealing, cultivates 18 days results.
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| CN105325300A (en) * | 2015-11-30 | 2016-02-17 | 华南农业大学 | Method of rapid propagation of distant hybridization progenies of Eriobotrya plants by embryo culture and application |
| CN105816368A (en) * | 2016-03-29 | 2016-08-03 | 福建省亚热带植物研究所 | Natural black-lighting, antibacterial and anti-inflammation scalp massage cream prepared from loquat cells |
| CN110710452A (en) * | 2019-11-05 | 2020-01-21 | 南京农业大学 | Tissue culture and rapid propagation method of eriobotrya japonica |
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| CN105816368A (en) * | 2016-03-29 | 2016-08-03 | 福建省亚热带植物研究所 | Natural black-lighting, antibacterial and anti-inflammation scalp massage cream prepared from loquat cells |
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