CN104885937A - Method for inducing Psammosilene tunicoides callus to produce anthocyanidin - Google Patents
Method for inducing Psammosilene tunicoides callus to produce anthocyanidin Download PDFInfo
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- CN104885937A CN104885937A CN201510247199.2A CN201510247199A CN104885937A CN 104885937 A CN104885937 A CN 104885937A CN 201510247199 A CN201510247199 A CN 201510247199A CN 104885937 A CN104885937 A CN 104885937A
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Abstract
The invention discloses a method for inducing Psammosilene tunicoides callus to produce anthocyanidin. The method includes: selecting the tender shoot of a Psammosilene tunicoides aseptic seedling as the explant, placing the explant in an MS basic medium, additionally adding 0.5mg/L 6-BA and 2.5mg/L IAA to induce callus; putting the induction produced white loose callus on the MS basic medium, adding 0.5mg/L 6-BA and 1.5mg/L NAA, and under the condition of illumination of 2000 lx, conducting culture to induce generation of anthocyanidin; and then subjecting the callus inducing anthocyanidin generation to subculture multiplication culture, taking MS as the basic medium, adding 1.0mg/L 6-BA, 0.5mg/L NAA and 0.3mg/L KT, thus finally obtaining the callus with high anthocyanidin content. The anthocyanidin produced by induction in the invention is a pure natural pigment with high nutrition and health care value and bright color.
Description
Technical field
The present invention relates to field of plant tissue culture technique, be specifically related to the regulate and control method of tuniclike psammosilene root callus induction and the formation of tuniclike psammosilene root callus anthocyan.
Background technology
Tuniclike psammosilene root (
psammosilene tunicoidesw.C.Wu et C.Y.Wu) have another name called RADIX PSAMMOSILENE, Sinense Knotweed Rhizome etc., belong to Caryophyllaceae tuniclike psammosilene root and belong to perennial herb, be mainly distributed in the ground such as China Guizhou, Yunnan, Sichuan, Tibet, grow in the gravel hillside of height above sea level 2000 ~ 3800 m or calcium carbonate rock seam.Tuniclike psammosilene root is a kind of Endangered Medicinal Herb; its root is used as medicine; there is the function such as scattered silt pain relieving, toxin expelling hemostasis; within 1991, be put into " Chinese Plants Red Data Book " first as rare endangered species, within 1999, be put in " national key protected wild plants register (first) " as Chinese Second Class Key Protected Plant.Tuniclike psammosilene root is longer in Yunnan Medicinal history, and the Chinese patent medicine preparation utilizing modern means of science and technology to develop has that longstalk condorvine herb is loose, fracture and pain-stop cream, golden bone lotus capsule etc.Tuniclike psammosilene root plants stems is many in purple green, and duration of flowering petal is aubergine, and namely the cell of tuniclike psammosilene root plant has the ability of synthesis of natural pigment, and therefore, while utilizing its root, the natural colouring matter of these species of comprehensive exploitation also has a extensive future.
Summary of the invention
The invention provides a kind of method of inducing tuniclike psammosilene root callus to produce anthocyan, enriched the kind of the natural plant pigment needed for people, also for the comprehensive development and utilization of this rare resources of tuniclike psammosilene root opens new way.
The technical solution used in the present invention: first with tuniclike psammosilene root aseptic seedling for explant induction produces callus, then by the adjustment of plant growth regulating substance concentration and intensity of illumination to optimize the condition of callus anthocyan formation.
Concrete steps are as follows:
Step 1: adopt tuniclike psammosilene root aseptic seedling as explant, be placed in MS medium, add 6-BA 0 ~ 3.0 mg/L and IAA 0 ~ 2.0 mg/L and cultivate, induction obtains callus;
Step 2: select loose or fine and close callus, take MS as minimal medium, add 6-BA 0 ~ 2.5 mg/L and NAA 0 ~ 2.5 mg/L and cultivate under illumination 0 ~ 2500 lx condition, after 7 d, white callus induces red cyanidin to generate;
Step 3: the callus containing anthocyan that selecting step 2 obtains carries out squamous subculture, take MS as minimal medium, additional 6-BA 1.0 mg/L+NAA 0.5 mg/L+KT 0.3 mg/L+2, and 4-D 0 ~ 2.0 mg/L cultivates, and after 30 d, anthocyan obviously accumulates;
Step 4: the callus of pigment accumulation under selection same culture conditions, take hydrochloric acid-methanol as extractant, under different solid-liquid ratios, different extraction times and different Extracting temperature, optimize pigment extraction conditions, and scan under 200 ~ 650 nm with ultraviolet specrophotometer.
Wherein, the condition of culture in step 1 is: temperature 24 ~ 26 DEG C, intensity of illumination 1000 lx, light application time 12 h/d.
The condition of culture of step 3 is: temperature 24 ~ 26 DEG C, intensity of illumination 2000 lx, light application time 12 h/d.
In step 4, the concentration of hydrochloric acid-methanol extractant is 1% ~ 3%.
Solid-liquid ratios different in step 4, different extraction times and different Extracting temperature refer to solid-liquid ratio between 1:10 ~ 1:50, extraction time between 5 ~ 20 h, Extracting temperature is between 4 ~ 50 DEG C.
Preferably, in step 1, the explant of evoked callus is the bud of tuniclike psammosilene root aseptic seedling, and the plant growth regulating substance of evoked callus is 6-BA 2.5 mg/L+IAA 0.5 mg/L, and cultivation temperature is 25 DEG C, intensity of illumination is 2000 lx, and light application time is 12 h/d.
Preferably, choose open-textured callus in step 2, the plant growth regulating substance of induction anthocyan is 6-BA 0.5 mg/L+NAA 1.5 mg/L.
Preferably, the intensity of illumination of inducing anthocyan to produce in step 2 is 2000 lx, and light application time is 12 h/d.
Preferably, in step 3, the condition of callus accumulation anthocyan is MS minimal medium additional 6-BA 1.0 mg/L, NAA 0.5 mg/L and KT 0.3 mg/L.
Preferably, in step 4, tuniclike psammosilene root callus anthocyan maximum absorption band is at n=530 nm, and pigment content is expressed as A530=Abs; Hydrochloric acid-methanol with 3% is extractant, solid-liquid ratio is 1:10, extraction time 20 h, Extracting temperature 30 DEG C time extract.
The beneficial effect that the present invention reaches:
(1) because the multipair health of synthetic food color exists certain harmfulness, and natural colouring matter has no side effect.The present invention is with the bud of tuniclike psammosilene root aseptic seedling for explant, and induction produces callus shoot proliferation, then selects the plant growth regulating substance that is suitable for and illumination condition, thus induces its callus to accumulate anthocyan.
(2) anthocyan that the present invention obtains is the biosynthetic natural colouring matter of tuniclike psammosilene root callus cell, is Flavonoid substances, has certain physiologically active, can be used for the industry such as food, medicine, to meet the demand of people to natural colouring matter.
Accompanying drawing explanation
Fig. 1 is the impact that different plant growth regulating substance combination (6-BA, NAA) is induced tuniclike psammosilene root callus anthocyan;
Fig. 2 is that different illumination intensity and 2,4-D are on the impact of tuniclike psammosilene root callus anthocyanin accumulation;
Fig. 3 is the ultra-violet absorption spectrum of tuniclike psammosilene root callus anthocyan.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Technical scheme concrete steps of the present invention are as follows:
(1) explant selects the induction with callus: adopt tuniclike psammosilene root aseptic seedling (stem, Ye Heya) as explant, be placed in MS medium, add 6-BA 0 ~ 3.0 mg/L and IAA 0 ~ 2.0 mg/L and cultivate under the condition of temperature (25 ± 1) DEG C, intensity of illumination 1000 lx and light application time 12 h/d, induction obtains callus;
(2) induction of anthocyan and accumulation: the callus selecting differing texture (loose or fine and close), take MS as minimal medium, add 6-BA 0 ~ 2.5 mg/L and NAA 0 ~ 2.5 mg/L, cultivate under illumination 0 ~ 2500 lx, after 7 d, white callus induces red cyanidin to generate; Select a small amount of callus containing anthocyan and carry out squamous subculture, take MS as minimal medium, additional 6-BA 1.0 mg/L+NAA 0.5 mg/L+KT 0.3 mg/L+2,4-D 0 ~ 2.0 mg/L is in temperature (25 ± 1) DEG C, cultivate under the condition of intensity of illumination 2000 lx and light application time 12 h/d, after 30 d, anthocyan obviously accumulates.
(3) extraction of tuniclike psammosilene root callus pigment: the callus (blotting callus surface moisture with filter paper) of pigment accumulation under selection same culture conditions, with 1% ~ 3% hydrochloric acid-methanol for extractant, at different solid-liquid ratios (1:10 ~ 1:50), different extraction time, (5 ~ 20 h) and under different Extracting temperature (4 ~ 50 DEG C) optimized pigment extraction conditions, and scan under 200 ~ 650 nm with ultraviolet specrophotometer.
Preferably, the explant of evoked callus is bud, and the Inducing plant growth Auto-regulator of callus is 6-BA 2.5 mg/L+IAA 0.5 mg/L, and cultivation temperature is (25 ± 1) DEG C, and intensity of illumination is 2000 lx, and light application time is 12 h/d.
Preferred, the callus chosen requires that quality is loosened, and it is 6-BA 0.5 mg/L+NAA 1.5 mg/L that induction anthocyan produces plant growth regulating substance used.As shown in table 1 and Fig. 1.In Fig. 1, A is white callus (6-BA 2.0 mg/L+NAA 1.0 mg/L); B is aubergine callus (6-BA 0.5 mg/L+NAA 1.0 mg/L); C is purple callus (6-BA 0.5 mg/L+NAA 1.5 mg/L)
Preferred, the illumination condition that induction anthocyan produces is for 2000 lx(are as shown in table 2 and Fig. 2).Preferred, the condition of callus anthocyanin accumulation for when not adding 2,4-D (as shown in table 2 and Fig. 2), additional 6-BA 1.0 mg/L, NAA 0.5 mg/L and KT 0.3 mg/L.
In Fig. 2, D, E, F, G, H, I represent different illumination intensity (500 lx, 1000 lx, 1500 lx, 2000 lx, the 2500 lx) influence degree to anthocyanin accumulation respectively, and when wherein intensity of illumination is 2000 lx, (H) pigment accumulation effect is best; J, K represent the impact of 2,4-D addition on pigment accumulation, (K) pigment accumulation successful when wherein not adding 2,4-D
In addition, as can be seen from Figure 3, tuniclike psammosilene root pigment maximum absorption band is at n=530 nm place, and pigment content can be expressed as A
530=Abs(Absorbance writes a Chinese character in simplified form, and represents absorbance); Hydrochloric acid-methanol with 3% is extractant, solid-liquid ratio is 1:10, extraction time 20 h, Extracting temperature 30 DEG C time extraction effect better.
Embodiment 1:
Vegetable material tuniclike psammosilene root in the present embodiment (
psammosilene tunicoides) pick up from prestige Ning County of Guizhou Province.
1. explant selects the induction with callus:
Using the aseptic seedling of tuniclike psammosilene root seed germination (stem, Ye Heya) as explant, because the differentiation and proliferation ability of bud is strong, the new young young stem and leaf formed is easy to induce and produces new callus, and propagation rapidly, therefore selects bud better as the explant material of evoked callus.
Diameter is selected to be the culture dish of 9 cm, take MS as minimal medium, add 6-BA 2.5 mg/L, IAA 0.5 mg/L, sucrose 30 g/L, agar 7 g/L, 5 buds inoculated by each culture dish, cultivation temperature is (25 ± 1) DEG C, and intensity of illumination is 2000 lx, and light application time is 12 h/d, produce callus after cultivating 7 d, 30 d Multiplying culture of follow-up generation are to obtain a large amount of callus.
2. the induction of anthocyan and accumulation:
The induction of anthocyan: because non embryogenic callus quality is loosened, cell is relatively large, includes large vacuole, and pigment is present in vacuole mostly, therefore must select the induction of open-textured white callus for anthocyan.Take 2.5 g white callus callus and be placed in culture dish respectively, take MS as minimal medium, additional 6-BA 0.5 mg/L, NAA 1.5 mg/L, sucrose 30 g/L, agar 7 g/L, cultivation temperature is (25 ± 1) DEG C, intensity of illumination is 2000 lx, and light application time is 12 h/d.
The accumulation of anthocyan: need to carry out squamous subculture to accumulate a large amount of anthocyan after tuniclike psammosilene root white callus produces a small amount of anthocyan.Take 3.0 g during squamous subculture and be placed in MS minimal medium with the callus of anthocyan, add 6-BA 1.0 mg/L, NAA0.5 mg/L, KT0.3 mg/L, sucrose 30 g/L, agar 7 g/L, cultivation temperature is (25 ± 1) DEG C, intensity of illumination is 2000 lx, light application time is 12 h/d, can make more than 90% Callus formation anthocyan after 30 ~ 40 d.
3. the extraction of tuniclike psammosilene root callus anthocyan:
Select there is the callus of anthocyanin accumulation (blotting callus surface moisture with filter paper), on average take 1 g callus to grind in mortar, adding 10 mL 3% hydrochloric acid-methanol is transferred in 25 mL test tubes, and 20 h are extracted in 30 DEG C of water-baths, be placed in n=530 nm place again and measure light absorption value, each process repeats 3 times.Calculate pigment recovery rate, pigment extraction rate reached 0.41%.
Recovery rate:
In formula:
afor pigment is in the absorbance at 530 nm wavelength places;
vfor constant volume;
nfor extension rate during colorimetric; 98.2 is the extinction coefficient of pigment at 530 nm wavelength places;
mfor callus quality.
embodiment 2:
the extraction optimization of tuniclike psammosilene root callus anthocyan:
The callus of anthocyanin accumulation (blotting callus surface moisture with filter paper) is had under selecting same culture conditions, on average take 1 g callus to grind in mortar, process according to selected four factors (volume fraction, solid-liquid ratio, time and temperature), the results are shown in Table 3.
As known from Table 3, solid-liquid ratio is main affecting factors, and along with solid-liquid ratio increases, light absorption value reduces to be because solid-liquid two-phase exists absorption and dissolution equilibrium, and low temperature contributes to absorption, because solid-liquid ratio increases, while the identical heat of generation, temperature reduces, and suction-operated strengthens, and absorbance reduces.
What the present invention adopted is that plant tissue culture technique produces natural plant pigment, and callus proliferation is fast, and pigment production is high, and is not subject to seasonal restrictions, and can meet the need of market in time.
Because natural colouring matter is bright in colour, have no side effect, and health-care efficacy is given prominence to, gradually by people are pursued, induce with short production cycle, speed fast by tissue culture technique, equipment investment is few in addition, extraction process is simple, and production cost is low, and Developing Prospects on Industrialized Exploitation is very wide.
The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications are also considered as the scope that the present invention protects.
Claims (10)
1. induce tuniclike psammosilene root callus to produce a method for anthocyan, it is characterized in that: with the aseptic seedling of tuniclike psammosilene root seed germination for material obtains callus through tissue cultures, by producing anthocyan to the adjustment evoked callus of condition of culture;
Concrete steps are as follows:
Step 1: adopt tuniclike psammosilene root aseptic seedling as explant, be placed in MS medium, add 6-BA 0 ~ 3.0 mg/L and IAA 0 ~ 2.0 mg/L and cultivate, induction obtains callus;
Step 2: select loose or fine and close callus, take MS as minimal medium, add 6-BA 0 ~ 2.5 mg/L and NAA 0 ~ 2.5 mg/L and cultivate under illumination 0 ~ 2500 lx condition, after 7 d, white callus induces red cyanidin to generate;
Step 3: the callus containing anthocyan that selecting step 2 obtains carries out squamous subculture, take MS as minimal medium, additional 6-BA 1.0 mg/L+NAA 0.5 mg/L+KT 0.3 mg/L+2,4-D 0 ~ 2.0 mg/L cultivates, and after 30 d, anthocyan obviously accumulates;
Step 4: the callus of pigment accumulation under selection same culture conditions, take hydrochloric acid-methanol as extractant, under different solid-liquid ratios, different extraction times and different Extracting temperature, optimize pigment extraction conditions, and scan under 200 ~ 650 nm with ultraviolet specrophotometer.
2. induction tuniclike psammosilene root callus according to claim 1 produces the method for anthocyan, it is characterized in that: the condition of culture in step 1 is: temperature 24 ~ 26 DEG C, intensity of illumination 1000 lx, light application time 12 h/d.
3. induction tuniclike psammosilene root callus according to claim 1 produces the method for anthocyan, it is characterized in that: the condition of culture of step 3 is: temperature 24 ~ 26 DEG C, intensity of illumination 2000 lx, light application time 12 h/d.
4. induction tuniclike psammosilene root callus according to claim 1 produces the method for anthocyan, it is characterized in that: the concentration of hydrochloric acid-methanol extractant is 1% ~ 3%.
5. induction tuniclike psammosilene root callus according to claim 1 produces the method for anthocyan, it is characterized in that: described different solid-liquid ratio, different extraction times and different Extracting temperature refer to solid-liquid ratio between 1:10 ~ 1:50, extraction time between 5 ~ 20 h, Extracting temperature is between 4 ~ 50 DEG C.
6. induction tuniclike psammosilene root callus according to claim 1 produces the method for anthocyan, it is characterized in that: in step 1, the explant of evoked callus is the bud of tuniclike psammosilene root aseptic seedling, the plant growth regulating substance of evoked callus is 6-BA 2.5 mg/L+IAA 0.5 mg/L, cultivation temperature is 25 DEG C, intensity of illumination is 2000 lx, and light application time is 12 h/d.
7. induction tuniclike psammosilene root callus according to claim 1 produces the method for anthocyan, it is characterized in that: choose open-textured callus in step 2, and the plant growth regulating substance of induction anthocyan is 6-BA 0.5 mg/L+NAA 1.5 mg/L.
8. induction tuniclike psammosilene root callus according to claim 1 produces the method for anthocyan, and it is characterized in that: the intensity of illumination of inducing anthocyan to produce in step 2 is 2000 lx, light application time is 12 h/d.
9. induction tuniclike psammosilene root callus according to claim 1 produces the method for anthocyan, it is characterized in that: in step 3, the condition of callus accumulation anthocyan is MS minimal medium additional 6-BA 1.0 mg/L, NAA 0.5 mg/L and KT 0.3 mg/L.
10. induction tuniclike psammosilene root callus according to claim 1 produces the method for anthocyan, it is characterized in that: in step 4, tuniclike psammosilene root callus anthocyan maximum absorption band is at n=530 nm, and pigment content is expressed as A
530=Abs; Hydrochloric acid-methanol with 3% is extractant, solid-liquid ratio is 1:10, extraction time 20 h, Extracting temperature 30 DEG C time extract.
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Cited By (5)
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| CN106244515A (en) * | 2016-08-04 | 2016-12-21 | 福建省农业科学院农业工程技术研究所 | A kind of method improving Vitis davidi cell culture anthocyanidin content |
| CN110305797A (en) * | 2019-06-18 | 2019-10-08 | 北京理工大学 | One plant of anthocyanidin producing bacterial strain CJ6 and its application |
| CN113796316A (en) * | 2021-10-18 | 2021-12-17 | 大连工业大学 | Culture medium for promoting psammosilene tunicoides hairy root callus to produce anthocyanin and induction method |
| CN113913362A (en) * | 2021-10-18 | 2022-01-11 | 大连工业大学 | Sorbus commixta stem cell capable of increasing anthocyanin content, culture medium and culture method |
| CN115812601A (en) * | 2022-12-15 | 2023-03-21 | 贵州慧创科技发展有限公司 | Method for inducing generation of anthocyanin by utilizing Psammosilene tunicoides hairy roots |
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| CN106244515A (en) * | 2016-08-04 | 2016-12-21 | 福建省农业科学院农业工程技术研究所 | A kind of method improving Vitis davidi cell culture anthocyanidin content |
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| CN113796316A (en) * | 2021-10-18 | 2021-12-17 | 大连工业大学 | Culture medium for promoting psammosilene tunicoides hairy root callus to produce anthocyanin and induction method |
| CN113913362A (en) * | 2021-10-18 | 2022-01-11 | 大连工业大学 | Sorbus commixta stem cell capable of increasing anthocyanin content, culture medium and culture method |
| CN115413580A (en) * | 2021-10-18 | 2022-12-02 | 大连工业大学 | Culture medium and culture method for promoting psammosilene tunicoides hairy root callus to generate anthocyanin |
| CN113913362B (en) * | 2021-10-18 | 2023-11-17 | 大连工业大学 | Sorbus pohuashanensis stem cell for improving anthocyanin content, culture medium and culture method |
| CN115812601A (en) * | 2022-12-15 | 2023-03-21 | 贵州慧创科技发展有限公司 | Method for inducing generation of anthocyanin by utilizing Psammosilene tunicoides hairy roots |
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